Truncation SELEX method

ABSTRACT

This invention is directed to a method for identifying nucleic acid ligands by the SELEX method wherein the participation of fixed sequences is eliminated or minimized.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/907,111, filed Jul. 17, 2001, now U.S. Pat. No. 6,855.496, which is acontinuation of U.S. patent application Ser. No. 09/275,850, filed Mar.24, 1999, now U.S. Pat. No. 6,261,774, which is a continuation-in-partof U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, nowU.S. Pat. No. 5,475,096, which is a continuation-in-part of U.S. patentapplication Ser. No. 07/536,428, filed Jun. 11, 1990, now abandoned.

FIELD OF THE INVENTION

The present invention is directed to methods for identifyingoligonucleotide sequences which specifically bind to target molecules.More particularly, this invention is directed to methods for identifyingoligonucleotide sequences in which the participation of fixed sequencesis eliminated or minimized.

BACKGROUND OF THE INVENTION

The dogma for many years was that nucleic acids had primarily aninformational role. Through a method known as Systematic Evolution ofLigands by Exponential enrichment, termed SELEX, it has become clearthat nucleic acids have three dimensional structural diversity notunlike proteins. SELEX is a method for the in vitro evolution of nucleicacid molecules with highly specific binding to target molecules and isdescribed in U.S. patent application Ser. No. 07/536,428, filed Jun. 11,1990, entitled “Systematic Evolution of Ligands by ExponentialEnrichment,” now abandoned, U.S. patent application Serial No.07/714,131, filed Jun. 10, 1991, entitled “Nucleic Acid Ligands.” nowU.S. Pat. No. 5,475,096 and U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled “Methods for Identifying NucleicAcid Ligands,” now U.S. Pat. No. 5,270,163 (see also WO 91/19813), eachof which is specifically incorporated by reference herein. Each of theseapplications, collectively referred to herein as the SELEX PatentApplications, describes a fundamentally novel method for making aNucleic Acid Ligand to any desired target molecule. The SELEX processprovides a class of products which are referred to as Nucleic AcidLigands, each ligand having a unique sequence, and which has theproperty of binding specifically to a desired target compound ormolecule. Each SELEX-identified Nucleic Acid Ligand is a specific ligandof a given target compound or molecule. SELEX is based on the uniqueinsight that Nucleic Acids have sufficient capacity for forming avariety of two- and three-dimensional structures and sufficient chemicalversatility available within their monomers to act as ligands (formspecific binding pairs) with virtually any chemical compound, whethermonomeric or polymeric. Molecules of any size or composition can serveas targets.

The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of Nucleic Acids, preferably comprising asegment of randomized sequence, the SELEX method includes steps ofcontacting the mixture with the target under conditions favorable forbinding, partitioning unbound Nucleic Acids from those Nucleic Acidswhich have bound specifically to target molecules, dissociating theNucleic Acid-target complexes, amplifying the Nucleic Acids dissociatedfrom the Nucleic Acid-target complexes to yield a ligand-enrichedmixture of Nucleic Acids, then reiterating the steps of binding,partitioning, dissociating and amplifying through as many cycles asdesired to yield highly specific high affinity Nucleic Acid Ligands tothe target molecule.

It has been recognized by the present inventors that the SELEX methoddemonstrates that Nucleic Acids as chemical compounds can form a widearray of shapes, sizes and configurations, and are capable of a farbroader repertoire of binding and other functions than those displayedby Nucleic Acids in biological systems.

The present inventors have recognized that SELEX or SELEX-like processescould be used to identify Nucleic Acids which can facilitate any chosenreaction in a manner similar to that in which Nucleic Acid Ligands canbe identified for any given target. In theory within a Candidate Mixtureof approximately 10¹³ to 10¹⁸ Nucleic Acids, the present inventorspostulate that at least one Nucleic Acid exists with the appropriateshape to facilitate each of a broad variety of physical and chemicalinteractions.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, entitled “Method for Selecting Nucleic Acids on theBasis of Structure.” now abandoned (see U.S. Pat. No. 5,707,796),describes the use of SELEX in conjunction with gel electrophoresis toselect Nucleic Acid molecules with specific structural characteristics,such as bent DNA. U.S. patent application Ser. No. 08/123,935, filedSep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,” nowabandoned (see U.S. Pat. No. 5,763,177), describes a SELEX based methodfor selecting Nucleic Acid Ligands containing photoreactive groupscapable of binding and/or photocrosslinking to and/or photoinactivatinga target molecule. U.S. patent application Ser. No. 08/134,028, filedOct. 7, 1993, entitled “High-Affinity Nucleic Acid Ligands ThatDiscriminate Between Theophylline and Caffeine,” now U.S. Pat. No.5,580,737, describes a method for identifying highly specific NucleicAcid Ligands able to discriminate between closely related molecules,which can be non-peptidic, termed Counter-SELEX. U.S. patent applicationSer. No. 08/143,564, filed Oct. 25, 1993, entitled “Systematic Evolutionof Ligands by Exponential Enrichment: Solution SELEX,” now U.S. Pat. No.5,567,588, describes a SELEX-based method which achieves highlyefficient partitioning between oligonucleotides having high and lowaffinity for a target molecule.

The SELEX method encompasses the identification of high-affinity NucleicAcid Ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in stability or improveddelivery characteristics. Examples of such modifications includechemical substitutions at the ribose and/or phosphate and/or basepositions. SELEX-identified Nucleic Acid Ligands containing modifiednucleotides are described in U.S. patent application Ser. No.08/117,991, filed Sep. 8, 1993, entitled “High Affinity Nucleic AcidLigands Containing Modified Nucleotides,” now U.S. Pat. No. 5,660,985,that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S.patent application Ser. No. 08/134,028, supra, describes highly specificNucleic Acid Ligands containing one or more nucleotides modified with2′-amino (2′-NH₂), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S.patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled“Novel Method of Preparation of Known and Novel 2′-Modified Nucleosidesby Intramolecular Nucleophilic Displacement,” now abandoned, describesoligonucleotides containing various 2′-modified pyrimidines. The SELEXmethod encompasses combining selected oligonucleotides with otherselected oligonucleotides and non-oligonucleotide functional units asdescribed in U.S. patent application Ser No. 08/284,063, filed Aug. 2,1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX,” now U.S. Pat. No. 5,637,459, and U.S.patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled“Systematic Evolution of Ligands by Exponential Enrichment: BlendedSELEX,” now U.S. Pat. No. 5,683,867, respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules.

The SELEX method further encompasses combining selected nucleic acidligands with lipophilic compounds or non-immunogenic, high molecularweight compounds in a diagnostic or therapeutic complex as described inU.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled“Nucleic Acid Ligand Complexes.” VEGF Nucleic Acid Ligands that areassociated with a Lipophilic Compound, such as diacyl glycerol ordialkyl glycerol, in a diagnostic or therapeutic complex are describedin U.S. patent application Ser. No. 08/739,109, filed Oct. 25, 1996,entitled “Vascular Endothelial Growth Factor (VEGF) Nucleic Acid LigandComplexes,” now U.S. Pat. No. 5,859,228. VEGF Nucleic Acid Ligands thatare associated with a Lipophilic Compound, such as a glycerol lipid, ora Non-Immunogenic, High Molecular Weight Compound, such as polyethyleneglycol, are further described in U.S. patent application Ser. No.08/897,351, filed July 21, 1997, entitled “Vascular Endothelial GrowthFactor (VEGF) Nucleic Acid Ligand Complexes.” VEGF Nucleic Acid Ligandsthat are associated with a non-immunogenic, high molecular weightcompound or lipophilic compound are also further described in WO98/18480, published May 7, 1998, entitled “Vascular Endothelial GrowthFactor (VEGF) Nucleic Acid Ligand Complexes.” Each of the abovedescribed patent applications that describe modifications of the basicSELEX procedure are specifically incorporated by reference herein intheir entirety.

In the SELEX process, generally the candidate mixture includes regionsof fixed sequences (i.e., each of the members of the candidate mixturecontains the same sequences in the same location) and regions ofrandomized sequence. The fixed sequences are usually selected for: a)assisting in the amplification steps; b) mimicking a sequence known tobind to the target; or c) enhancing the concentration of a givenstructural arrangement of the nucleic acids in the candidate mixture.The fixed region(s) (or part of the fixed region) in theSELEX-identified Nucleic Acid Ligand may participate in binding to thetarget. Additionally, the fixed region(s) (or part of the fixed region)could form a structure(s) or contribute to a structure that binds to orfacilitates binding to the target. Although in some circumstances thisis a desirable attribute, in other circumstances the fixed region(s) maylimit the possible structural variation and number of different nucleicacid ligands resulting from the SELEX process. The development of amethod for generating nucleic acid ligands in which the participation offixed sequences in binding to the target is minimized or eliminated isdesirable.

Toole et al. (WO 92/14843) discloses a method for identifyingoligonucleotide sequences which specifically bind biomolecules. In oneembodiment, the method includes identifying and amplifyingoligonucleotides without attached flanking regions or structuralconstraints, but which nevertheless are capable of specific binding todesired targets. This method provides the ability to engineerappropriate means for amplifying the desired oligonucleotides. In thismethod, a pool of oligonucleotides is generated in which the sequencesare unknown. These sequences are incubated with the target underconditions wherein some of the oligonucleotides complex with the target.The oligonucleotides that complex with the target are recovered andknown sequences are added to at least one end of the oligonucleotide.These known sequences are then used in amplifying the nucleic acidligands. Once the nucleic acid ligands have been amplified, the knownnucleotide sequence is removed. The process may be repeated for thedesired number of rounds until an optimal nucleic acid ligand populationmay be identified.

SUMMARY OF THE INVENTION

The present invention describes methods for generating nucleic acidligands in which the participation of fixed sequences in binding to thetarget is minimized or eliminated.

In one embodiment of the method of this invention, a method forgenerating nucleic acid ligands without the participation of fixedsequences is described, comprising:

a) preparing a candidate mixture of single-stranded nucleic acidswherein each nucleic acid member of said candidate mixture comprises afixed region;

b) annealing oligonucleotides to the fixed sequences that arecomplementary to said fixed sequences;

c) contacting said candidate mixture with said target molecule;

d) partitioning the nucleic acids having, an increased affinity to thetarget molecule relative to the candidate mixture from the remainder ofthe candidate mixture; and

e) amplifying the increased affinity nucleic acids, in vitro, to yield aligand-enriched mixture of nucleic acids whereby nucleic acid ligands ofthe target molecule are identified.

In another embodiment of the method of this invention, a method forgenerating nucleic acid ligands without the participation of fixedsequences is described, comprising

a) preparing a candidate mixture of single-stranded nucleic acidswherein each nucleic acid member of said candidate mixture comprises oneor more regions of fixed sequences;

b) contacting said candidate mixture with said target molecule;

c) partitioning the nucleic acids having an increased affinity to thetarget molecule relative to the candidate mixture from the remainder ofthe candidate mixture;

d) amplifying the increased affinity nucleic acids, in vitro, to yield aligand-enriched mixture of nucleic acids;

f) replacing said one or more regions of fixed sequences with adifferent one or more regions of fixed sequences; and

g) repeating steps b)-d), whereby nucleic acid ligands of the targetmolecule are identified.

In yet another embodiment of the invention, a method for generatingnucleic acid ligands without the participation of fixed sequences isdescribed as follows:

a) contacting a candidate mixture with a target molecule;

b) partitioning the nucleic acids having an increased affinity to thetarget molecule relative to the candidate mixture from the remainder ofthe candidate mixture;

c) hybridizing the nucleic acids partitioned in step b) with a libraryof single stranded nucleic acids that are complementary to the singlestranded nucleic acids of the candidate mixture, wherein each nucleicacid member of the complementary library has a fixed region;

d) amplifying the nucleic acids that hybridized to a nucleic acid in thecomplementary library whereby increased affinity nucleic acid ligands ofthe target molecule having fixed regions are produced; and

e) cleaving said fixed regions of the increased affinity nucleic acidswhereby nucleic acid ligands of the target molecule are identified.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the strategy of changing the fixed sequences. See Example 1for detailed explanation.

FIGS. 2A-E show MS2 CP binding sites. FIG. 2A shows the consensusbinding site of MS2 CP. NN′ is any base pair, R is either A or G, Y iseither U or C. FIG. 2B shows a frequent selection artifact. The fixedsequence is shown in lowercase, the genomic insert in uppercase, thetail is underlined. FIG. 2C shows the actual genomic sequence (fromGenBank) that corresponds to the artifact shown in FIG. 2B. It is shown“folded” only for comparison with FIG. 2A and FIG. 2B, and is notpredicted to bind MS2 CP. FIG. 2D shows the predicted structure of themajor isolate (rffG) from SELEX with changing the fixed sequences. Thegenomic insert nucleotides are in uppercase, fixed sequence (startingwith ggg at the 5′ end) in lowercase. The consensus binding site isshown in boldface. FIG. 2E shows the SELEX consensus binding site of MS2CP. SS′ is either GC or CG base pair. The first 2 NN′ base pairs musthave at least one SS′.

FIG. 3 shows an outline of the genomic SELEX experiments 1-5, with thenumber of isolates sequenced at the end of each SELEX.

FIG. 4 shows binding of RNA to MS2 CP. SELEX isolates with the consensusbinding site, rffG and ebgR, bind well. To test whether the fixedsequences contribute to binding of the rffG isolate, the RNA fragmentthat corresponds exactly to its insert was obtained from E. coli genomicDNA using PCR and in vitro transcription. This RNA fragment (rffG minusfixed sequences) binds only marginally worse than the original rffGSELEX isolate. For comparison, the natural MS2 CP binding site(bacteriophage MS2 replicase fragment) binds more weakly than all of theSELEX isolates with the consensus binding site (Table 2), except secYisolate (data not shown). A typical SELEX isolate without the consensusbinding site does not appreciably bind MS2 CP, and neither does thestarting library (data not shown).

FIGS. 5A and B show the genomic sequence of rffG in the vicinity of theMS2 CP binding site. FIG. 5A shows the distribution of library 3′end-points in the sequence of rffG. I designates an end-point, which isthe terminal nucleotide of the genomic insert, right next to the libraryfixed sequence. The number of I's above a position is equal to thenumber of sequenced isolates (out of 19 total) with the end-point atthis position. The MS2 CP consensus binding site is underlined.Asterisks (*'s) designate the predicted range of end-points, given thesize of the genomic inserts of the library (65 nucleotides), andallowing room for binding both of the rffG genomic primers during theisolation of these molecules from the starting library. Thedouble-underlined sequence designates end-points that correspond to themajority (93%) of the IfsC isolates in SELEX. The positions of theseend-points were inferred from the isolates' sequences and from the FokIcutting specificity (9-13 nucleotides away from the FokI recognitionsite). The majority of the library end-points (I's) fall in the sameregion as the majority of end-points of the rffG SELEX isolates. FIG. 5Bshows a hypothetical event during library construction that may havecaused the observed clustering of end-points. The rffG genomic sequence(shown in FIG. 5A) is predicted to anneal weakly in its single-strandedform to the primer used in library construction. The primer has 9randomized nucleotides at the 3′ end, and a fixed 5′ end sequence. Thisfixed sequence anneals to some genomic sites, such as the one shown,better than to others, and thus may cause an over-representation of thecorresponding DNA in the library.

FIGS. 6A-C show the comparison of the truncation SELEX protocols toregular SELEX.

FIG. 7 shows the truncation SELEX by ligation process. Symbols are asfollows: T7P, T7 promoter sequence; 5′Fx, 5′ fixed sequence for 5′primer binding; 3′Fx, 3′ fixed sequence for 3′ primer binding; Rdm,random region; P5 cleav, chimeric oligonucleotide used for site directedcleavage at the 5′ fixed sequence by RNaseH; P3cleav, chimericoligonucleotide used for site directed cleavage at the 3′ fixed sequenceby RNaseH; T7PcI, DNA oligoniucleotide complementary to T7promoter-initiation sequence, annealed to a complementary DNAoligonucleotide extended by 5 random nucleotides; T7Pc2. DNAoligonucleotide complementary to T7 promoter sequence, annealed to acomplementary DNA oligonucleotide extended by the initiator sequence5′GGGA.

FIG. 8 shows truncation SELEX by tailing. Schematic representation ofthe truncation process by tailing is described. Symbols TdT, RT-RNaseH,T7P and FL, indicate terminal deoxyribonucleotide transferase, RNaseHactivity of reverse transcriptase, T7 promoter, and full length,respectively.

FIG. 9 shows truncation SELEX by hybrid selection process. Symbols areas follows: T7P, T7 promoter sequence; 5′Fx, 5′ fixed sequence for 5′primer binding; 3′Fx, 3′ fixed sequence for 3′ primer binding; Rdm,random region; P5 cleav, chimeric oligonucleotide used for site directedcleavage at the 5′ fixed sequence by RNaseH; P3cleav, chimericoligonucleotide used for site directed cleavage at the 3′ fixed sequenceby RNaseH; B, biotin.

FIG. 10 shows truncation SELEX by size selection at the DNA level. Anadvanced SELEX pool is PCR amplified using, primers lacking the T7promoter sequence. The PCR products are partially digested with DNaseIand size fractionated by gel electrophoresis. Size fractionated partialdigestion products are ligated to a plasmid digested by blunt endrestriction enzyme. Ligation products are then PCR amplified using a setof primers binding to plasmid sequences flanking the digestion site. Oneof the primers contains the sequence for the T7 promoter which may ormay not be part of the plasmid sequence. Such ligation reaction willcreate circular molecules having both possible orientations for eachinsert. To eliminate the molecules with the wrong orientation withrespect to the T7 promoter, transcripts from the final PCR library arehybridized with excess biotinylated transcripts from the startinglibrary and the hybrids are removed by streptavidin capture. Remainingunhybridized RNAs are then PCR amplified to generate the starting poolfor truncation SELEX by size selection.

FIG. 11 shows the schematic of the process of determining the 5′, 3′ orminimum binding fragment of a given Nucleic Acid Ligand. A hypotheticalsequence of an octamer is shown as full length and all possiblefragments resulting from partial digestion at each position. Thefragments that will be visible on a sequencing gel along with theexpected gel pattern are as shown at each labeling scheme. The patternof the T1 partial digestion (at G positions as shown by arrowheads (>))is also shown at the right side of each gel pattern. The body labeledfragment is assumed to be labeled at A positions. Symbols TB, AH, T1,represent target bound fragments, partial alkaline hydrolysis ladder,and partial RNaseT1 digestion ladder, respectively. The expectedtetramer minimum fragment is shown by arrow.

FIGS. 12A and B show the determination of minimum binding fragments ofseveral KGF ligand. Body labeled 2′F pyrimidine modified transcriptswere alkaline hydrolyzed in 50 mM Na₂CO₃, pH 9.0, 1 mM EDTA, at 90° C.for 10 minutes. Alkaline hydrolyzed RNA was recovered by ethanolprecipitation and was incubated with KGF in PBS, 0.01% HAS for 15 min at25° C. and bound RNA was partitioned by nitrocellulose filtration. Theamount of KGF used for each ligand was determined by the ligand's Kd andranged from 2-0.1 nM. Bound RNA was extracted from the nitrocellulosefilter with phenol urea and was analyzed on a sequencing gel. RNA fromcontrol reactions lacking KGF was also run for some ligands. Symbols56F, 38F, 14F, 53F, 26F, and 15F are designations for KGF ligands (KGFpatent); + or − indicate the use of KGF or not, respectively; M aremolecular markers with the indicated sizes. Minimum fragments are shownby arrowheads.

FIG. 13 shows the reconstruction experiment for determining the averageminimum fragments of pools. KGF ligand K14F 2′F transcript was mixedwith 40N7 at the indicated ratios. RNA mixes were digested with RNaseP1at 0.0024 U/pmole of RNA in manufacturers buffer at 65° C. for 5minutes. Prior to digestion the RNA were denatured in digestion buffercontaining 6.8 M urea (final). Digestion was done in the presence ofurea. Following digestion the RNA was recovered by ethanol precipitationand was phosphatased and 5′ end labeled by kinase as described. KinasedRNA was recovered by ethanol precipitation, then it was incubated withKGF at 200 pM and RNA:protein ratio of 100 at 25° C. for 15 minutes inPBS, 0.01% HSA and target bound RNA was recovered by nitrocellulosefiltration followed by urea/phenol recovery and ethanol precipitation.Nitrocellulose recovered RNA was analyzed on a sequencing gel. As acontrol, ligand K14F was partially alkaline hydrolyzed as described, andbound fragments were recovered and analyzed on the same gel. Minimumfragments are shown by arrowheads. Symbols AH, P1, L, +, − indicatealkaline hydrolysis, RNaseP1 digestion, starting RNA digest (ladder),incubation with KGF and incubation without KGF, respectively.

FIG. 14 shows partial digestion of 2′F pyrimidine modified RNA. Bodylabeled 30N7 2′F pyrimidine modified RNA was digested with RNaseP1 (P1),nuclease S7 (S7) or phosphodiesterase (PD). Digestions were done inmanufacturers specified buffers containing 7 M urea at 65° C. for 15minutes. RNA was present at 0.9 μM. Digests were analyzed on asequencing gel. Phosphodiesterase was used at 20, 2, 1, and 0.5 ng.Nuclease S7 was used at 3, 0.3, 0.03, and 0.003 units. RNaseP1 was usedat 0.1, 0.01, 0.001, and 0.0001 units. Lanes 1, 6, and 11 wereincubation reactions lacking enzyme.

FIG. 15 shows compounds used to biotinylate RNA at its 3′ end using T4RNA ligase.

FIG. 16 shows the digestion of 2′F pyrimidine modified transcripts byRNase H. Two enzymes were used as shown. The presence or absence of the5′ or 3′ targeting oligonucleotides are indicated by +and −,respectively. The sequence of the RNA and the targeting oligonucleotidesused are also shown.

FIG. 17 shows the mapping of RNaseH digestion sites using end labeledsubstrates. Two different Nucleic Acid Ligands were used in thisexperiment as described. Digestion sites are indicated by arrowheads.The alignment of the alkaline hydrolysis pattern with the RNA sequenceis as shown. The sequence of the RNA molecules and the digestion sitesare also shown under the gel patterns. Symbols FL, AH, 5′TR, and 3′TRindicate full length, alkaline hydrolysis, 5′ truncate and 3′ truncate.

FIG. 18 shows the mapping of RNaseH of the 5′ digestion site usingprimer extension. Two different Nucleic Acid Ligands were used as fulllength (FL) or 5′truncates (5′TR) in reverse transcriptase reactions inthe presence of 3G7 5′end labeled primer. RT reactions were done in thepresence or all 4 dNTPs (PE) or with one of ddATP (A), ddCTP (C), ddGTP(G), and ddTTP (T). Chain termination products were used to align thegel pattern to the sequence of the RNA used. The sequence of the RNAmolecules and the digestion sites (arrowheads) are also shown under thegel patterns.

FIG. 19 shows the digestion activity of various enzyme preparations onbody labeled 2′F pyrimidine modified RNA using the targetingoligonucleotides described. Enzymatic reactions were set as described.The presence or absence of the targeting oligonucleotides or the enzymesample is indicated by + or − respectively. Symbol FL, SD and DD,indicate full length, single digestion product, and double digestionproduct, respectively.

FIG. 20 shows the biotinylation efficiency of 2′F pyrimidine modifiedRNA by terminal transferase. Biotinylation reactions were set with 0.1mM biotin-dUTP and various amounts of body labeled RNA as shown, in 10μl reactions. Following incubation at 37° C. for 1 hour the reactionswere spotted on SAM filters (streptavidin loaded nitrocellulose filters,Promega, Madison, Wis.), washed 5 times with 2 ml of 50 mM NaCl, 10 mMTris-HCl pH 7.5, 1 mM EDTA and quantitated on an instant imager. Filterretained radioactivity was normalized to radioactivity loaded on filterswithout washing. Symbols B-NB and B-B indicate background on filterswithout or with preblocking, respectively. Background is defined as theamount of RNA from terminal transferase reactions lacking biotin-dUTPretained on the filters after washing.

FIG. 21 shows specificity of the hybrid selection step of the truncationSELEX by hybridization process. Hybrid selection of purified ssDNAcomplementary strands using terminal transferase biotinylated truncateRNA. VEGF VT30 Rd 12 RNA was RNaseH digested to remove 5′ and 3′ fixedregions and biotinylated using terminal transferase and biotin-dUTP.Biotinylated truncate RNA (0.3 pmoles) was mixed with 10 fold excess,5′-end-labeled ssDNA complementary strands from the same pool and washybridized overnight at 72° C. in the presence of 0.1 mM C-TAB in 10 mMTris, pH 7.5, 50 mM NaCl, 1 mM EDTA. Reactions with competitor salmonsperm DNA at 1-, 10-, 100-, and 1000-x over biotinylated RNA were alsoincluded. The RNA, ssDNA, and salmon sperm DNA were denatured at 95° C.and fast cooled prior to incubation at 72° C. Following hybridization,the reaction products were treated with streptavidin (+) or not (−) andanalyzed on a 10% acrylamide TBE gel. C indicates the position of thestreptavidin/hybridization reaction complex.

FIGS. 22A-C show binding properties of TGF∃1 pools with and withouttheir fixed sequences. Body labeled 2′F pyrimidine modified transcriptsfrom TGF∃1 40N7 round 12 (R12) (FIG. 22A), round 13 (R13) (FIG. 22B),and round 16 (R16) (FIG. 22C) were used as full length or werepreviously treated with RNaseH to remove the 5′ and 3′ fixed sequencesas described. The RNA was used to determine the fraction of RNA bound atvarious concentrations of TGF∃1 using nitrocellulose filter binding.Symbols ∘ and ● indicate full length and truncated RNA, respectively.

FIG. 23 shows binding properties of the Rd12 VEGF VT30 pool with andwithout its fixed sequences. Body labeled 2′F pyrimidine modifiedtranscripts were used as full length or were previously treated withRNaseH to remove the 5′ and 3′ fixed sequences as described. The RNA wasused to determine the fraction of RNA bound at various concentrations ofVEGF using nitrocellulose filter binding. Symbols ∘ and ● indicate fulllength and truncated RNA, respectively.

FIGS. 24A-C show the application of gel shifts to partition thehybridization products during the truncation SELEX by hybridizationprocess. In FIG. 24A selected truncate RNA from the first VEGF round ofthe truncation SELEX by hybridization was hybridized to full lengthcomplementary strands as described and the hybridization products weretreated (+SA) or not (−SA) and then electrophoresed on native 10%polyacrylamide TBE gel. The streptavidin/hybrid complex (SA Hyb-C) fromboth the +SA and −SA lane were excised and recovered as described. FIG.24B shows PCR amplification products at the indicated number of cyclesusing the eluted material from the +SA and −SA gel slices. FIG. 24Cshows selected truncate RNA from the second VEGF round of the truncationSELEX by hybridization was hybridized to full length complementarystrands as described and the hybridization products were partitioned asabove. For the second round both the template generated by the +SA(designated Rd1) and the −SA (designated Rd1-ct) material were used asshown.

FIG. 25 shows the effectiveness of hybrid selection using streptavidinbeads. Hybridization reactions between biotinylated truncated RNAtranscripts and full length antisense ssDNA were set as described.Control reactions were also set in the absence of biotinylated RNA.Hybrids were captured and recovered with streptavidin beads asdescribed. Captured ssDNA was PCR amplified and the product was analyzedby gel electrophoresis at different PCR cycles as shown. The gel wascalibrated using a 20 bp ladder.

FIG. 26 shows the effect of removal of fixed sequences on the affinitiesof ligands from the starting, selected, and control libraries obtainedin the VEGF truncation SELEX by hybridization experiment. Full lengthand truncated RNA transcripts from individual ligands were used todetermine Kd and plateau values using nitrocellulose filter bindingcurves. Each graph point is defined by the Kd and plateau for an RNA.Open circles are affinity values obtained with full length RNA whileclosed circles are affinity values from truncated RNA (lacking both 5′and 3′ fixed sequences). The plots were separated into four quadrants byplacing all the full length points in quadrant I.

FIG. 27 shows the frequency of affinity points in the four affinityquadrants from the VEGF truncation SELEX by hybridization experiment.

FIG. 28 shows nitrocellulose filter binding curves with a set of ligandsfrom the TGF∃1 40N truncation SELEX by the hybridization experiment. AllRNAs were high specific activity body labeled and were digested toremove their fixed regions. RNA was incubated with variousconcentrations of TGF∃1 and bound RNA was partitioned by nitrocellulosefiltration and quantitated. Ligands tested are as shown. The binding oftruncated random RNA (rd0) is also shown.

FIGS. 29A-C show PCR using short primers. The sequence of the template,primers, and expected reaction products are as shown in FIG. 29A. PCR1indicates (FIG. 29B) the gel results with PCR reactions at twotemperatures in the presence (+) or absence (−) of 10% DMSO using thetemplate and primers shown in the top. PCR2 indicates (FIG. 29C) the gelresults for RT/PCR reactions starting with 0.5 pmole of RNA transcriptgenerated using the template from PCR1. The RT/PCR product was analyzedfollowing 10 or 20 PCR cycles. The gels were calibrated using 20base-pair marker.

FIG. 30 shows the electrophoretic analysis of RT/PCR products using the30N7 and the 25NTR template-primer set. 1 pmole of starting RNA wasdiluted 10-fold or 5-fold as shown and was subject to RT/PCR in thepresence of appropriate primers as described. The products following 15PCR cycles were analyzed on a 10% polyacrylamide TBE gel. The identityand size of the products are as indicated. Heterodublex representsannealed products having nonhomologous random regions. The size in basesof the markers used are as shown.

FIG. 31 shows the bioactivity of TGF∃1 Nucleic Acid Ligands isolatedfrom the truncation SELEX by hybridization. RNA was synthesized byphosphoramidite chemistry. Indicator cells (mink lung epithelial cells)were incubated with TGF∃1 and dilutions of RNA as described. The extentof cell proliferation was measured by ³H-thymidine incorporation and thedata were analyzed as described. The points represent an average of n=2and error bars are standard errors. The sequence of CD70syn and30N7-9syn are 5′GGGUGCCUUUUGCCUAGGUUGUGAUUUGUAACCUUCUGCCCA (SEQ IDNO:323) and 5′AGGGGUCUGGAAUUUUUGGUUUACCCGUACGCU (SEQ ID NO:324),respectively.

FIG. 32 shows the generation of ssDNA for truncation SELEX with DNAlibraries. The left and middle branch show processes to generate sensestrands of the library lacking the fixed sequences.

The right branch shows the process to generate the antisense full lengthstrand of the library for the hybridization SELEX process.

FIGS. 33A and B show biotinylation of 2′F pyrimidine modified RNA withT4 RNA ligase. In FIG. 33A RNA ligase reactions were set that includedthe critical components as shown under each lane. dCB and dCCBBB werekinased in the presence of α-³²P-ATP prior to use. Reactions shown inlanes 1-8 were done at 4° C. in the presence of 2-fold excess of dCB ordCCBBB over RNA. Reactions in lanes 9-14 were done at 0° C. in thepresence of 20-fold excess of dCB or dCCBBB over RNA. LP, and 40-03designate ligated products and the use of TGF∃1 Nucleic Acid Ligand40-03 described in U.S. application Ser. No. 09/046,247, filed Mar. 23.1998 (which is incorporated herein in its entirety), respectively. FIG.33B shows streptavidin gel shifts of gel purified ligated products fromthe gel depicted in FIG. 33A. Purified ligated products and the use ofstreptavidin is as shown under each lane.

FIGS. 34A and B show the ligation of the 3′ fixed sequence at the 3′ endof 2′F pyrimidine modified RNA. FIG. 34A depicts ligation products fromreactions containing body labeled RNA transcripts from the indicatedtemplates and the critical components as shown were analyzed on a 10%acrylamide, 8 M urea gel. Symbols SC, LP and TR indicate streptavidincomplexes, ligated products, and unligated transcript, respectively.FIG. 34B depicts the sequence of the two templates used to generate RNAtranscripts for this experiment. Arrowheads indicated the site ofcleavage of restriction sites as follows: The 35N-Nae template containsdownstream of the random region the site 5′GGCCGGCC which is digestedsymmetrically by the restriction enzyme FseI between the second andthird base from the 3′ end. The FseI site contains the restriction sitefor NaeI (5′GCCGGC) which is digested by NaeI symmetrically between thethird and fourth base from the 5′ end. The 35N-Bsm template containsdownstream of the random region the site 5′GCATTC which is digestedasymmetrically by the restriction enzyme BsmI as shown. The RNAtranscripts derived from restriction digested templates are as shown.

FIGS. 35A-D show the ligation of the 5′ fixed sequence. FIGS. 35 A and Bshow at the 5′ end of 2′F pyrimidine modified RNA. Schematic of thereaction reactants and products are shown in FIG. 35A. Results from theligation reaction at the 5′ end of RNA is shown in FIG. 35B. Thecritical components of the reaction are as shown. FIGS. 35C and D showligation of the 5′ fixed sequence at the 3′ end of cDNA. The schematicof the reaction reactants and products are shown in FIG. 35C. Two setsof ligation oligo/bridge oligo were used as shown. The results from theligation reaction at the 3′ end of cDNA is shown in FIG. 35D. (I)Results with the 5G7RC Linker/5′N7 oligonucleotides. (II) Results withthe 5N7 Linker/5N7 Bridge oligonucleotides. The ratio of linkeroligonucleotide to cDNA is shown above each lane. Symbol LC indicatesligated products.

FIGS. 36A and B show properties of pools from the VEGF truncation SELEXby ligation experiment. FIG. 36A depicts the size of the PCR productsfrom the starting round 12 VT30 pool, starting 5′ truncated pool (LTR0),truncation SELEX round 1 (LTR1) and truncation SELEX round 2 (LTR2). Thesize of molecular weight markers is as shown. FIG. 36B depictsproperties of the 5′ truncated starting pool. Body labeled 2′Fpyrimidine modified transcript was digested with RNaseH to remove the 3′fixed sequence. Digested 5′ truncated starting pool (LTR0) and digestedat both ends random RNA (30N.Rd0) were used to determine the fraction ofRNA bound at various concentrations of VEGF using nitrocellulose filterbinding. Circles and squares indicate LTR0 and 30N.Rd-0, respectively.

FIGS. 37A-F show nitrocellulose filter binding curves with an exampleset of ligands from the VEGF round 1 truncation SELEX by ligationexperiment. All RNAs were high specific activity body labeled. Bindingwas measured with undigested and RNaseH truncated (designated by T afterthe ligand designation) RNA lacking both the bulk of 5′ and the 3′ fixedsequences. RNA was incubated with various concentrations of VEGF andbound RNA was partitioned by nitrocellulose filtration and quantitated.Ligands tested are as shown.

FIGS. 38A-C show the effect of removal of fixed sequences on theaffinities of ligands from the starting 5′ truncated (5′TR) (FIG. 38A),round 1 (Rd1) (FIG. 38B), and round 2 (Rd2) (FIG. 38C) librariesobtained in the VEGF truncation SELEX by ligation experiment. Fulllength and truncated RNA transcripts from individual ligands were usedto determine Kd and plateau values using nitrocellulose filter bindingcurves. Each graph point is defined by the Kd and plateau for an RNA.Open circles are affinity values obtained with starting full lengthround 12 VT30 RNA. Closed circles are affinity values from truncated RNA(lacking both the bulk of 5′ and the 3′ fixed sequences). Open squaresare affinity values from ligands containing their 3′ fixed sequences butlacking the bulk of their 5′ fixed sequence. The closed circles and opensquares come from the same set of ligands within each library shown,while the open circles are a different set, coming from the round 12VT30 pool. This different set of VT30 ligands was used to definequadrant I. Quadrant IV of the starting 5′TR pool contained 10-13 pointswith Kd>1×10⁻⁶ M and plateaus <5%. These points were set as Kd=1×10⁻⁶and plateau=5.

FIG. 39 shows the frequency of affinity points in the four affinityquadrants from the VEGF truncation SELEX by ligation experiment.

FIGS. 40A and B show nitrocellulose filter binding curves with a set ofligands from the TGF∃1 30N and 40N truncation SELEX by ligationexperiment. All RNAs were high specific activity body labeled and weredigested to remove their fixed regions. RNA was incubated with variousconcentrations of TGF∃1 and bound RNA was partitioned by nitrocellulosefiltration and quantitated. Ligands tested are as shown. The binding oftruncated 40-03 ligand (described in U.S. patent application Ser. No.09/046,247, filed Mar. 23, 1998) is also shown.

FIGS. 41A and B show the enzymatic addition of the fixed sequences toselected truncated ssDNA from truncation SELEX with DNA libraries. FIG.41A shows the schematic addition of the fixed sequences by ligationusing appropriately designed stem-loop structures. FIG. 41B shows theproposed sequences for the process shown in FIG. 41A. P indicates theaddition of 5′ phosphates during phosphoramidite synthesis.

FIG. 42 shows the comparison of the progress of the VEGF truncationSELEX by hybridization and ligation experiments. During the SELEXrounds, the amount of RNA bound to the target was quantitated andexpressed as % fraction of expected max binding and was plotted as afunction of target concentration as shown. Pools shown are: ligation 5′truncated starting pool (LRT0), ligation round 1 (LRT1), ligation round2 (LRT2). VT30 round 12 (HRT0), hybridization round 1 (HRT1),hybridization round 1 control (HART1-ct), hybridization round 2 (HRT2),and hybridization round 2 control (HRT2-ct).

FIGS. 43A and B show the comparison of phenotype frequencies fromligands obtained in the truncation SELEX by hybridization and ligationas shown. For phenotype determination, the binding of each ligand wasdetermined with and without its fixed sequence and then each ligand wasevaluated if it retained binding following removal of its fixedsequence. Phenotypes were determined by filter binding and wereclassified according to five groups as follows: (1) molecules that losesignificant affinity for VEGF upon removal of their fixed sequences; (2)molecules with affinities for VEGF somewhat affected (positively ornegatively) upon removal of their fixed sequences; (3) molecules thatgain significant affinity for VEGF upon removal of their fixedsequences; (4) molecules with affinities for VEGF not affected uponremoval of their fixed sequences; and (5) non-binding ligands eitherwith or without fixed sequences.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

“Nucleic Acid Ligand” or “Aptamer” as used herein is a non-naturallyoccurring Nucleic Acid having a desirable action on a Target. Adesirable action includes, but is not limited to, binding of the Target,catalytically changing the Target, reacting with the Target in a waywhich modifies/alters the Target or the functional activity of theTarget, covalently attaching to the Target as in a suicide inhibitor,facilitating the reaction between the Target and another molecule. Inthe preferred embodiment, the action is specific binding affinity for aTarget molecule, such Target molecule being a three dimensional chemicalstructure other than a polynucleotide that binds to the Nucleic AcidLigand through a mechanism which predominantly depends on Watson/Crickbase pairing or triple helix binding, wherein the Nucleic Acid Ligand isnot a Nucleic Acid having the known physiological function of beingbound by the Target molecule.

“Candidate Mixture” is a mixture of Nucleic Acids of differing sequencefrom which to select a desired ligand. The source of a Candidate Mixturecan be from naturally occurring Nucleic Acids or fragments thereof,chemically synthesized Nucleic Acids, enzymatically synthesized NucleicAcids or Nucleic Acids made by a combination of the foregoingtechniques. In a preferred embodiment, each Nucleic Acid has fixedsequences surrounding a randomized region to facilitate theamplification process.

“Nucleic Acid” means either DNA, RNA, single-stranded or double-strandedand any chemical modifications thereof. Modifications include, but arenot limited to, those that provide other chemical groups thatincorporate additional charge, polarizability, hydrogen bonding,electrostatic interaction, and fluxionality to the Nucleic Acid Ligandbases or to the Nucleic Acid Ligand as a whole. Such modificationsinclude, but are not limited to, 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil, backbone modifications such asintemucleoside phosphorothioate linkages, methylations, unusualbase-pairing combinations such as the isobases isocytidine andisoguanidine and the like. Modifications can also include 3′ and 5′modifications such as capping.

“SELEX” methodology involves the combination of selection of NucleicAcid Ligands that interact with a Target in a desirable manner, forexample binding to a protein, with amplification of those selectedNucleic Acids. Iterative cycling of the selection/amplification stepsallows selection of one or a small number of Nucleic Acids that interactmost strongly with the Target from a pool which contains a very largenumber of Nucleic Acids. Cycling of the selection/amplificationprocedure is continued until a selected goal is achieved. The SELEXmethodology is described in the SELEX Patent Applications. The SELEXmethodology is sometimes referred to herein as Conventional SELEX.

“Genomic SELEX” is a variation on the SELEX methodology in which thenucleic acids in the randomized region of the candidate mixture arereplaced by genomic sequences (or inserts) derived from an organism.Genomic SELEX is also sometimes referred to herein as Conventional (orRegular) Genomic SELEX when the method is performed without changing thefixed sequences or annealing of oligonucleotides. It will be understoodfrom the context of the specification whether Genomic SELEX is beingperformed with or without changing the fixed sequences or annealing ofoligonucleotides.

“Truncation SELEX” is a variation on the SELEX methodology in which theparticipation of fixed sequences in the binding to the Target isminimized or eliminated.

“Truncation SELEX by Ligation” is a variation on the Truncation SELEXMethod whereby amplifiable molecules are created by introducing fixedregions with a ligation reaction after interaction with the target(i.e., the nucleic acid does not contain fixed sequences when itinteracts with the target). An example of Truncation SELEX by Ligationis illustrated in FIG. 7. Other examples of the method are alsocontemplated.

“Truncation SELEX by Hybridization” is a variation on the TruncationSELEX Method whereby amplifiable molecules are obtained by hybridizingthe selected nucleic acid (containing minimal or no fixed sequences) tothe original candidate mixture. An example of Truncation SELEX byHybridization is illustrated in FIG. 9. Other examples of the method arealso contemplated.

“Target” means any compound or molecule of interest for which a ligandis desired. A Target can be a protein (such as PDGF, thrombin, andselectin), peptide, carbohydrate, polysaccharide, glycoprotein, hormone,receptor, antigen, antibody, virus, substrate, metabolite, transitionstate analog, cofactor, inhibitor, drug, dye, nutrient, growth factor,etc. without limitation.

The SELEX process provides a class of products which are Nucleic Acidmolecules, each having a unique sequence, and each of which has theproperty of binding specifically to a desired Target compound ormolecule. Target molecules are preferably proteins, but can also includeamong others carbohydrates, peptidoglycans and a variety of smallmolecules. SELEX methodology can also be used to Target biologicalstructures, such as cell surfaces or viruses, through specificinteraction with a molecule that is an integral part of that biologicalstructure.

In its most basic form, the SELEX process may be defined by thefollowing series of steps:

1) A Candidate Mixture of Nucleic Acids of differing sequence isprepared. The Candidate Mixture generally includes regions of fixedsequences (i.e., each of the members of the Candidate Mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the Target, or (c) to enhance the concentration of agiven structural arrangement of the Nucleic Acids in the CandidateMixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

2) The Candidate Mixture is contacted with the selected Target underconditions favorable for binding between the Target and members of theCandidate Mixture. Under these circumstances, the interaction betweenthe Target and the Nucleic Acids of the Candidate Mixture can beconsidered as forming Nucleic Acid-target pairs between the Target andthose Nucleic Acids having the strongest affinity for the Target.

3) The Nucleic Acids with the highest affinity for the target arepartitioned from those Nucleic Acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of Nucleic Acid) corresponding to the highest affinityNucleic Acids exist in the Candidate Mixture, it is generally desirableto set the partitioning criteria so that a significant amount of theNucleic Acids in the Candidate Mixture (approximately 5-50%) areretained during partitioning.

4) Those Nucleic Acids selected during partitioning as having therelatively higher affinity for the target are then amplified to create anew Candidate Mixture that is enriched in Nucleic Acids having arelatively higher affinity for the target.

5) By repeating the partitioning and amplifying steps above, the newlyformed Candidate Mixture contains fewer and fewer unique sequences, andthe average degree of affinity of the Nucleic Acids to the target willgenerally increase. Taken to its extreme, the SELEX process will yield aCandidate Mixture containing one or a small number of unique NucleicAcids representing those Nucleic Acids from the original CandidateMixture having the highest affinity to the target molecule.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, entitled “Method for Selecting Nucleic Acids on theBasis of Structure,” now abandoned (see U.S. Pat. No. 5,707,796),describes the use of SELEX in conjunction with gel electrophoresis toselect Nucleic Acid molecules with specific structural characteristics,such as bent DNA. U.S. patent application Ser. No. 08/123.935, filedSep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,” nowabandoned (see U.S. Pat. No. 5,763,177), describes a SELEX based methodfor selecting Nucleic Acid Ligands containing photoreactive groupscapable of binding and/or photocrosslinking to and/or photoinactivatinga target molecule. U.S. patent application Ser. No.08/134,028, filedOct. 7, 1993, entitled “High-Affinity Nucleic Acid Ligands ThatDiscriminate Between Theophylline and Caffeine,” now U.S. Pat. No.5,580,737, describes a method for identifying highly specific NucleicAcid Ligands able to discriminate between closely related molecules,termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filedOct. 25, 1993, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Solution SELEX,” now U.S. Pat. No. 5,567,588, describes aSELEX-based method which achieves highly efficient partitioning betweenoligonucleotides having high and low affinity for a target molecule.U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992,entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev.” now U.S. Pat.No. 5,496,938, describes methods for obtaining improved Nucleic AcidLigands after SELEX has been performed. U.S. patent application Ser. No.08/400,440, filed Mar. 8, 1995, entitled “Systematic Evolution ofLigands by Exponential Enrichment: Chemi-SELEX,” now U.S. Pat. No.5,705,337, describes methods for covalently linking a ligand to itstarget.

The SELEX method encompasses the identification of high-affinity NucleicAcid Ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified Nucleic Acid Ligands containingmodified nucleotides are described in U.S. patent application Ser. No.08/117,991, filed Sep. 8, 1993, entitled “High Affinity Nucleic AcidLigands Containing Modified Nucleotides.” now U.S. Pat. No. 5,660,985,that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S.patent application Ser. No. 08/134,028, supra, describes highly specificNucleic Acid Ligands containing one or more nucleotides modified with2′-amino (2′-NH₂), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S.patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled“Novel Method of Preparation of Known and Novel 2′ Modified Nucleosidesby Intramolecular Nucleophilic Displacement,” describes oligonucleotidescontaining various 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. patent application Ser. No. 08/284,063, filed Aug.2, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX,” now U.S. Pat. No. 5,637,459, and U.S.patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled“Systematic Evolution of Ligands by Exponential Enrichment: BlendedSELEX,” now U.S. Pat. No. 5,683,867, respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules.

The SELEX method further encompasses combining selected Nucleic AcidLigands with Lipophilic Compounds or Non-Immunogenic, High MolecularWeight Compounds in a diagnostic or therapeutic Complex as described inU.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled“Nucleic Acid Ligand Complexes.” The SELEX method further encompassescombining selected VEGF Nucleic Acid Ligands with lipophilic compounds,such as diacyl glycerol or dialkyl glycerol, as described in U.S. patentapplication Ser. No. 08/739,109, filed Oct. 25, 1996, entitled “VascularEndothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes,” nowU.S. Pat. No. 5,859,229. VEGF Nucleic Acid Ligands that are associatedwith a High Molecular Weight, Non-Immunogenic Compound, such asPolyethylene glycol, or a Lipophilic Compound, such as Glycerolipid,phospholipid, or glycerol amide lipid, in a diagnostic or therapeuticcomplex are described in U.S. patent application Ser. No. 08/897,351,filed Jul. 21, 1997, entitled “Vascular Endothelial Growth Factor (VEGF)Nucleic Acid Ligand Complexes.” Each of the above described patentapplications that describe modifications of the basic SELEX procedureare specifically incorporated by reference herein in their entirety.

SELEX identifies Nucleic Acid Ligands that are able to bind targets withhigh affinity and with outstanding specificity, which represents asingular achievement that is unprecedented in the field of Nucleic Acidsresearch. These characteristics are, of course, the desired propertiesone skilled in the art would seek in a therapeutic or diagnostic ligand.

In order to produce Nucleic Acid Ligands desirable for use as apharmaceutical, it is preferred that the Nucleic Acid Ligand (1) bindsto the target in a manner capable of achieving the desired effect on thetarget; (2) be as small as possible to obtain the desired effect; (3) beas stable as possible; and (4) be a specific ligand to the chosentarget. In most situations, it is preferred that the Nucleic Acid Ligandhas the highest possible affinity to the target. Additionally, NucleicAcid Ligands can have facilitating properties.

In commonly assigned U.S. patent application Ser. No. 07/964,624, filedOct. 21, 1992, now U.S. Pat. No. 5,496,938 ('938), methods are describedfor obtaining improved Nucleic Acid Ligands after SELEX has beenperformed. The '938 patent, entitled “Nucleic Acid Ligands to HIV-RT andHIV-1 Rev,” is specifically incorporated herein by reference.

As discussed above, generally the candidate mixture in the SELEX processincludes regions of fixed sequences and randomized sequences. The fixedsequences are usually present for assisting in the amplification stepsof the SELEX process and may participate in binding to the target orcontribute to structures that bind to the target. In some circumstances,the participation of the fixed sequences in binding to the target maynot be desirable. Generation of nucleic acid ligands in which theparticipation of fixed sequences in binding to the target is minimizedor eliminated can be accomplished is several ways:

1) The participation of fixed regions in the binding of the target couldbe reduced or eliminated by annealing complementary oligonucleotides tothe fixed regions prior to contacting the candidate mixture with thetarget. The double-stranded region is then bound together throughWatson/Crick base pairing, and, therefore, is not available for bindingto the target.

2) The fixed regions can also be changed at different rounds. As thesesequences are changed, their participation of any one fixed region inbinding is reduced.

3) Reduce the size of the fixed regions.

4) Eliminate the fixed sequences before interaction with the target.

As described above, in some circumstances it is desirable to have shortligands following the SELEX process. The reduction in the fixed regionof the template will accomplish reduction in the overall size of theindividual molecules. Reduction of the fixed regions is limited in orderto maintain PCR amplification. The shortest primer binding site reportedfor specific amplification is 7-bases long (Vincent et al. (1994) DNAand Cell Biology 13:75-82). Based on this approach, templates weredesigned to allow amplification of RNA that has 5′ and 3′fixed regionsof 5- and 8-bases long (FIG. 29). RT/PCR efficiency of such abbreviatedtemplate is about two logs lower than the N7 template series butnevertheless, sub-picomole amounts of RNA can be amplified within areasonable number of PCR cycles (FIG. 30).

Reduction of the random region of the template will also accomplishreduction in the overall size of the individual molecules. Random regionof a certain length however might be crucial in the isolation of highaffinity and bioactivity ligands as evident by experiments wherelibraries with random regions of different length were applied to thesame target in parallel. In one such experiment where TGF∃1 was theSELEX target, it was determined that the length of the random regionaffected the recovery of bioactive ligands with only the 40N librariesyielded high affinity bioactive ligands, as described in U.S. patentapplication Ser. No. 09/046,247, filed Mar. 23, 1998.

To further illustrate the approach for elimination of fixed sequences,the primer binding sites are removed from the molecules of the library,before the interaction with the target. After selection, primer bindingsites are re-introduced for amplification and generation of the pool forthe next round. Primer-building-site-removal requires nucleolyticdigestion either before or after generation of the population of singlestranded molecules. In SELEX experiments requiring transcription,removal of at least the 5′ fixed region must occur after transcriptionbecause digestion before transcription will remove the promoter site andwill eliminate transcription.

For SELEX experiments using DNA based libraries, removal of primerbinding sites can be done as described in FIG. 32. The originalsynthetic oligonucleotide library utilized for this approach can beengineered to include restriction endonuclease digestion sites thatgenerated 3′ or 5′ recessive ends as shown in FIG. 32. For digestionafter generation of single stranded molecules, oligonucleotidescomplementary to the fixed regions are annealed and the complexes aredigested by appropriate restriction endonucleases. Alternatively,digestion can occur at the dsDNA level and digested single strands canbe purified by incorporation of biotin at the 3′ recessive restrictionsite by using the Klenow fragment of E. coli DNA polymerase-I andbiotin-dUTP. For biotinylation at one end, restriction digestion at thatend can be followed by a fill-in reaction with the Klenow DNApolymerase-I when a restriction enzyme that generates 3′ recessive sitesis used, or by a terminal transferase reaction in the presence ofbiotin-dUTP. Biotinylation reactions are then followed by restrictiondigestion at the other end. If the digestion occurs at the ssDNA levelthe use of 3′ versus 5′ recessive restriction sites is not important andany restriction site can be used.

For SELEX experiments using RNA based libraries, removal of primerbinding sites can be done by site specific digestion by RNaseH asdescribed below, where DNA-2′ OMeRNA oligonucleotides are annealed toRNA molecules and the hybridization products are digested by RNaseH.

Following digestion and selection the fixed sequences need to be addedback to the library in order to allow amplification and carry the poolforward to the next round. Generation of amplifiable pools followingnuclease digestion and affinity selection can be achieved as describedbelow in two ways (FIG. 6B and 6C), namely either enzymatically(Truncation-SELEX-by-Ligation or Truncation-SELEX-by-Tailing) or byhybrid selection of purified complementary strands from the pool in use(Truncation-SELEX-by-Hybrid-Selection).

Overview of Truncation-SELEX-by-Ligation

This is an enzymatic method of Truncation SELEX. The steps ofTruncation-SELEX-by-ligation are summarized in FIG. 7. This approach canbe applied either at the beginning of a SELEX experiment or followingseveral regular rounds of the SELEX process.

For de novo Truncation-SELEX-by-ligation, the starting syntheticoligonucleotide pools can be designed to include just the random region.In order to avoid potential counterselection pressure due totranscription efficiency, especially if modified nucleotides are used aspolymerase substrates, it might be beneficial to include a transcriptioninitiation sequence immediately downstream of the T7 promoter such as5′GGGAG followed by the random region. The starting pool may include a3′ primer binding site or it may not. If no 3′ primer binding site isused at the starting pool, then single stranded oligonucleotides can beconverted to double stranded templates by including the T7promoter-initiator complement at the 3′ end of the random region, forexample 5′[N]₃₀CTCCCTATAGTGAGTCGTATTA (SEQ ID NO:325), and performingprimer extension using a primer such as 5′TAATACGACTCACTATAGGGAG (SEQ IDNO:46). If there is a 3′ primer binding site, then RNasell digestion canbe used, as described below, to remove that fixed sequence beforebinding to the target. Alternatively, a starting pool of DNA templatescan be used where appropriate restriction sites were incorporateddownstream of the T7 promoter and random region as shown in FIG. 41. Thepool of RNA is then generated by transcription of templated digested bythe appropriate restriction enzyme.

For application of truncation SELEX on pools evolved by conventionalSELEX experiments, the primer binding sites are removed by digestion andthen they are reintroduced following affinity selection by ligation.These steps will be done at each truncation SELEX round. Alternatively,a 5′ truncated library can be generated before affinity selectionstarts. This is a preferential alternative to avoid the use of a randombridge sequence for the 5′ end ligation since such ligation condition isinefficient. The generation of the 5′ truncated library will allowintroduction of the transcription initiation sequence 5′GGGAG adjacentto the 5′ end of the random region of the template. This initiationsequence in addition of relieving potential transcription selectionbias, it can also provide a specific sequence for the ligation bridgeused in the introduction of the 5′ primer binding site. Therefore, aconventional SELEX pool (FIG. 7-1) is converted into dsDNA template byextension with the 3′ primer; the ds template is then transcribed togenerate the full length RNA transcripts (FIG. 7-2); these transcriptsare then digested with RNaseH to remove either both or just the 5′primerbinding site (FIG. 7-3); the digested RNA is then reverse transcribed(FIG. 7-5) using the 3′primer after ligation of the 3′primer bindingsite by T4 RNA ligase (if necessary (FIG. 7-4)) to generate appropriatecDNA (FIG. 7-6); the generated cDNA is then ligated using T4 DNA ligase(FIG. 7-7) to the DNA oligonucleotide encoding the T7 promoter-initiator(5′TAATACGACTCACTATAGGGAGNNNNN (SEQ ID NO:37)) which has beenpreannealed to the bridge DNA oligonucleotide 5′CTCCCTATAGTGAGTCGTATTA(SEQ ID NO:36) (Table 4, set two); the ligation product (FIG. 7-8) isPCR amplified to generate a new dsDNA template (FIG. 7-9) lacking thebulk of the 5′ primer binding site; this new template is transcribed togenerate a 5′ truncated transcript (FIG. 7-10); the 5′ truncated RNA isthen digested with RNaseH at the 3′ end (FIG. 7-11) to generate thetruncated RNA for selection (FIG. 7-12).

For each truncation-SELEX-by-ligation round, truncated RNA (FIG. 7-13)generated by either method described above is bound to the target, andbound molecules are partitioned with an appropriate partition method andligated to the 3′ primer binding site (FIG. 7-14) using RNA ligase andan appropriate DNA oligo complementary to the 3′primer. The resultingligation product is reverse transcribed using the 3′ primer (FIG. 7-15)to generate cDNA (FIG. 7-16). The cDNA is then ligated (FIG. 7-17) tothe DNA oligonucleotide encoding the T7 promoter (5′TAATACGACTCACTATAGGGA (SEQ ID NO:35)) which has been preannealed to the bridge DNAoligonucleotide 5′TATAGTGAGTCGTATTA (SEQ ID NO:34) (Table 4, set one),and PCR amplified (FIG. 7-18) to generate a new dsDNA template (FIG.7-19); this new template pool is then transcribed to generate a new RNApool which is carried to the next round following removal of the 3′primer binding site by RNaseH digestion (FIG. 7-20).

Overview of Truncation SELEX by Tailing

This is an alternative enzymatic method of Truncation SELEX. The stepsof Truncation-SELEX-by-tailing are summarized in FIG. 8. This approachis similar to the truncation SELEX by ligation protocol where the fixedsequences are the result of a tailing reaction by the terminal deoxytransferase instead of ligase. Like the ligation approach, it can beapplied either at the beginning of a SELEX experiment or followingseveral rounds of the regular SELEX process. According to this scheme,selected truncated RNA (generated as described in the ligationprotocol), (FIG. 8-1) is incubated with terminal deoxy transferase (TdT)and a single dNTP to add homopolymeric tail at its 3′ end (FIG. 8-2).The length of the tail can be adjusted by including either ddNTP or NTPsat different ratios to the dNTPs to slow down the tailing reaction(Schmidt et al. (1996) Nucleic Acids Research 24:1789-1791). Tailed RNAis then reverse transcribed with reverse transcriptase (RT) and acomplementary oligonucleotide to the added tail (FIG. 8-3). Thecomplementary to the tail oligonucleotide could be engineered to containa couple of fixed positions to its 3′ end complementary to fixedpositions of the 3′ end of the truncated RNA so it can be anchoredadjacent to the 3′ end of the selected RNA. The generated cDNA (FIG.8-4) is then tailed with TdT and a different dNTP (FIG. 8-5) preferablywith pyrimidines to generate templates with appropriate T7 transcriptioninitiation sites. The generated tailed cDNA can be amplified by PCR(FIG. 8-6) using the 3′ primer used in the RT reaction and a 5′ primerengineered to be complementary to the 3′ tail of the cDNA and to containthe T7 promoter sequence to allow in vitro transcription of the selectedand PCR amplified pool (FIG. 8-7) for the next SELEX cycle. The in vitrotranscribed transcript (FIG. 8-8) can then be digested at both ends withRNaseH and appropriate targeting 2-OMe-deoxy oligonucleotide chimeras togenerated the new truncated RNA pool (FIG. 8-9) for the next round ofselection.

Overview of Truncation-SELEX-by-Hybrid-Selection

The steps of RNA Truncation-SELEX-by-hybrid-selection are summarized inFIG. 9. This approach relies in the ability of selected RNA to functionas a capture probe of its complementary sequence found in the same poolthat generated the RNA used in this particular round. Complementarysequences are generated by either strand separation and purificationfrom the dsDNA PCR product (Pagratis (1996) Nucleic Acids Research24:3645-6) or by in vitro T3 or SP6 transcription from templatesdesigned to contain appropriate promoter sequences at the 5′ end of thenegative strands as part of the 3′ fixed sequence. Such complementarystrands are still full length, containing their primer binding sites andtherefore PCR amplifiable. This method can be applied to both RNA andDNA SELEX differing only by the method of generation of single strandedpool and the digestion of such single stranded pool. For DNA SELEX, thesingle stranded molecules are generated by strand separation (Pagratis(1996) Nucleic Acids Research 24:3645-6) and are digested as describedabove. For RNA SELEX, the single stranded molecules are generated by invitro transcription followed by RNaseH digestion.

Each RNA truncation-SELEX-by-hybrid-selection round, starts with a dsDNAtemplate (FIG. 9-1) generated by PCR in the presence of 5′ primerbiotinylated at its 5′ end. A portion of the dsDNA template is used topurify the template (negative) strand as described above, while anotherportion is used to generate the single stranded selection pool, forexample RNA (FIG. 9-2); the single stranded selection pool is thendigested, for example with RNaseH (FIG. 9-3); digested molecules (FIG.9-4) are partitioned based on their binding to the target (FIG. 9-5);partitioned molecules are biotinylated, for example with terminaldeoxynucleotidyl transferase (FIG. 9-6); biotinylated selected moleculesare then hybridized to prepurified template strands from the samestarting pool and hybrids are partitioned by streptavidin capture (FIG.9-7); captured template strands are then PCR amplified (FIG. 9-8) togenerate the new pool for the next truncation-SELEX-by-hybrid-selectionround.

When the hybrid selection method is used, the hybridization rate ofcomplex nucleic acids must be taken into consideration, so enoughhybridization time is given at early rounds to allow capture of selectedsequences. There is a practical limit on the complexity of pools thatcan be utilized in the hybrid selection method. This practical limitneeds to be determined experimentally in the presence of compounds thatenhance hybridization rate such as CTAB (Pontius and Berg (1991) Proc.Natl. Acad. Sci. U.S.A. 88:8237-8241; Nedbal et al. (1997) Biochemistry36:13552-57).

Size Selection

This approach is a variation of the scheme, which involves removal offixed sequence and subsequent reintroduction of the same or differentfixed sequences either enzymatically, or by hybrid selection. Itinvolves the random fragmentation of an advanced or semiadvanced SELEXpool at either the DNA or RNA level followed by size selection (forexample using gel electrophoresis), and finally by introduction of fixedsequences either enzymatically or by hybrid selection.

Digestion at the DNA level (FIG. 10) can be done with DNaseI andfragments of specified length are extracted from a sizing gel slice (orby any other method i.e. density gradient sedimentation, gel filtrationetc.) and ligated to a circular vector (for example PCR-Script,Stratagene, Inc., La Jolla, Calif.). Ligation reactions are then servedas templates for PCR amplification using a new set of fixed sequences,which include the T7 RNA polymerase promoter with the 5′, fixedsequence. PCR amplification of the pool can follow E. colitransformation but this step could be limiting in sequence space due tothe E. coli transformation efficiency (10¹⁸-10¹⁰). The PCR generatedpool is the starting library of the truncation SELEX where the pool istranscribed by T7 RNA polymerase, digested by RNaseH to remove the newfixed sequences, and affinity selected by binding to the target.Selected RNA is then amplified by reintroducing fixed sequences byeither the truncation SELEX-by-Ligation, -Tailing or -Hybrid Selectionmethods as described above.

Digestion at the RNA level can be done with a nuclease and digested RNAcan be size selected by gel electrophoresis (or any other method). Sizeselected RNA can either be used for affinity selection directly or canbe used to generate a starting pool for subsequent truncation SELEXrounds. When RNA is used directly following affinity selection, the nextpool is generated by either the truncation SELEX-by-Ligation, -Tailingor -Hybrid Selection methods as described above. When RNA is used togenerate the starting pool for truncation SELEX, fixed sequences can beintroduced enzymatically by either the truncation SELEX-by-Ligation, or-Tailing methods as described above. The new pools are then used asdescribed above.

Screening for Ligands of a Certain Size

There is a desire for obtaining short ligands following application ofthe SELEX process for economical reasons. Therefore an efficient way toscreen for ligands that can be truncated to an appropriate length couldbe very useful. Screening can be done by procedures that allow boundarydetermination. Currently boundary determination (Fitzwater and Polisky(1996) Methods Enzymol 267:275-301) is done in two separate experimentswhere each boundary is first determined separately followed byexperiments where both boundaries are tested together in one moleculefor binding activity (FIG. 11). This procedure is tedious and it can bedone in a small set of ligands. Furthermore, there are examples thatcombination of the two boundaries, determined independently in the samemolecule, results in inactive ligands (data not shown).

Presented here is an alternative way of screening for ligands that cantolerate truncation to a given length. This approach is based on randomcleavage of internally labeled RNA ligands and then partition of thosefragments that retain binding to the target followed by gelelectrophoresis (FIG. 11). Random cleavage of oligonucleotides on nlength generates (n(n+1))/2 fragments (FIG. 11). Among these fragmentsonly those that contain the minimum necessary fragment will retainbinding to the target. Electrophoretic analysis on sequencing gels ofall randomly generated fragments that have been partitioned for targetbinding will generate a pattern containing a continuous range of partiallength fragments greater or equal of a minimum length (FIG. 11). Thismethod can be used to screen ligands for their possible minimal length.

Therefore, the size of the minimal fragment necessary for binding can bedetermined in a large number of ligands at once as follows. Each ligandis randomly cleaved to generate a ladder of molecules. These moleculesare radioactively labeled by either using body labeled starting materialor end labeling the resulting fragments, following fragmentation. Endlabeling can be done with either kinase or RNA ligase or terminaltransferase with γ-³²P-ATP or ³²P-pCp or α³²P-ddNTP, respectively. Theresulting radiolabeled ladder is allowed to bind to the target and thetarget bound molecules are then partitioned and analyzed on a sequencinggel along with size markers. Following autoradiography, minimum retainedfragment for each ligand can be observed and ligands with minimumfragments of desired length can be identified.

In addition to screening, minimum fragment methodology can be used fortruncation SELEX where the minimum fragments are excised from the geland used in the truncation SELEX schemes described above.

EXAMPLE 1 Experimental Procedures for Performing the SELEX Process byAnnealing of the Complementary Oligonucleotides to the Fixed SequenceRegion or by Changing the Fixed Sequences

This example provides general procedures followed by and incorporatedinto Example 2.

Materials and Methods

Genomic Library

The library from E. coli B genomic DNA was constructed as describedpreviously (Singer et al. (1997) Nuc. Acids Res. 25:781-786). Briefly,genomic DNA was denatured and annealed to a primer with a fixed 5′ endand 9 randomized nucleotides at the 3′ end. After annealing at 2° C.,the primer was extended with Klenow on ice, followed by roomtemperature, and 50° C. Another primer with a different fixed sequencewas added, and the denaturation/annealing/extension was repeated. Themolecules were separated by size on a denaturing polyacrylamide gel, andamplified by PCR using the above two primers minus the randomizedsequences, plus the T7 promoter.

Genomic SELEX

A non-aggregating MS2 CP variant V75E;A81G with RNA-binding propertiesidentical to wild type was purified as described previously (LeCuyer etal. (1995) Biochemistry 34:10600-6). Any endogenous E. coli RNA wasremoved in the purification process, as indicated by the bindingstoichiometry and the UV absorbance spectra (LeCuyer et al. (1995)Biochemistry 34:10600-6).

SELEX was initiated with 1 nmole of RNA, transcribed from the E. coligenomic DNA library. This amount is theoretically equivalent to morethan 10⁷ copies of every possible genomic insert, assuming the insertsstart with equal probability at any position within the E. coli genome.

In each round of selection, 1 nmole of gel-purified RNA (a mixture ofunlabeled RNA and a trace amount of RNA labeled during transcriptionwith [α-³²P]GTP) was denatured in TE at 95° C. for 1 minute, quicklychilled on ice and incubated on ice for 10 more minutes. Binding bufferwas added to give a final concentration of 100 mM HEPES-KOH, pH 7.5, 80mM KCl, 10 mM MgCl₂. The mixture was pre-filtered through nitrocellulose(0.45 micron pore, 25 mm diameter filter unit, Micro Filtration Systems(Dublin, Calif.), connected to a 3 ml disposable syringe and pre-wettedwith 0.5 ml of the binding buffer) to reduce the fraction of thenitrocellulose-binding RNA. The volume was adjusted with the bindingbuffer to make up for the loss on the filter. MS2 CP was added to thefinal concentration of 100 nM of the dimer in a 0.1 ml reaction. Bindingproceeded for 45 minutes at room temperature (22-24° C.).

The binding reaction was vacuum manifold-filtered through nitrocellulose(0.45 micron pore, 25 mm diameter, Micron Separations, Westborough,Mass.) and washed with 5 ml of the binding buffer. The fraction of thebound RNA, that is retained on nitrocellulose, was estimated by Cerenkovcounting. The protein and RNA concentrations were chosen so that inevery round this fraction was as low as possible, usually less than 1-2%of the total (to speed up the selection), but higher than in the controlreaction without MS2 CP (to reduce the “background” selection ofnitrocellulose binders).

The bound RNA was eluted from the cut filters by denaturation at 95° C.for 2 minutes in a suspension of 0.5 ml of 8 M urea in TBE and 0.5 ml ofphenol, and then amplified for the next round of SELEX essentially as inTuerk ((1997) Methods Mol. Biol. 67:219-230) with the following changes.RNA and primer B (5′-tcccgctcgtcg tctg-′ (SEQ ID NO:3)) were denaturedat 95° C. for 1 minute, annealed at 70° C. for 10 minutes, and reversetranscribed at 48° C. for 30 minutes with SuperScript (Gibco BRL)reverse transcriptase (these temperatures were chosen to melt RNAsecondary structures). The cDNA was amplified in PCR with Taq DNApolymerase as in Singer et al. ((1997) Nuc. Acids Res. 25:781-786) withprimers B and A (5′-gaaattaatacgactcactatagggaggacgatgcgg-3′ (SEQ IDNO:326); T7 promoter underlined). In this PCR, as well as in all othersin this study, the relatively low concentrations of MgCl₂ (3 mM) anddNTPs (50:M each) served to decrease the error rate. RNA wastranscribed, labeled and gel-purified as in Schneider et al. ((1992) J.Mol. Bio. 228:862-9) and Tuerk ((1997) Methods Mol. Biol. 67:219-230).After the completion of SELEX. DNA was cloned and sequenced as in Singeret al. ((1997) Nuc. Acids Res. 25:781-786).

SELEX with Annealing of the Complementary Oligonucleotides

Instead of 1 nmole of RNA, 0.1 nmoles of RNA and 0.4 nmoles of each ofthe two complementary oligonucleotides were used (Table 1). An extra 10minute incubation at room temperature was introduced directly after theaddition of the binding buffer to allow oligonucleotides to anneal. Theannealed oligonucleotides decreased the yield of the full-length reversetranscription product only by 10%. Otherwise, this SELEX was identicalto the conventional SELEX.

SELEX with Changing the Fixed Sequences

Step 1. Changing, the 3′ fixed sequence: DNA purification and PCR.Conventional SELEX was carried out for 3 rounds using the old fixedsequence primers A and B described above. Since the old fixed sequencesdid not contain FokI restriction sites, the sites had to be introducedby PCR (alternatively, the sites can be introduced during the libraryconstruction). DNA product from either the reverse transcriptionreaction, or from PCR after round 3, was purified from primer B. PrimerB interferes with the subsequent steps if not completely removed.Reverse transcription product was purified on a Microcon-30 filter(Amicon, Mass.), by centrifugation 3 times with 0.2 ml of TE buffer for10 minutes at 16,000×g. PCR product, since it contains more primer B,had to be purified, instead of Microcon-30, by native polyacrylamide gelelectrophoresis (PAGE) with ethidium bromide staining, followed bycrush-and-soak elution for 30 minutes at 37° C.

Purified DNA was amplified by PCR (FIG. 1) using primer A and primerB+FokI (5′-tcccgctcgtGgATGg-3′ (SEQ ID NO:327)). Primer B+FokIintroduces the FokI recognition site (GgATG, shown in boldface) into theold 3′ fixed sequence, and differs from primer B at the uppercasenucleotides (G, ATG).

The amplified DNA was extracted with chloroform, phenol, and 2 moretimes with chloroform, then ethanol precipitated and resuspended inwater (unpurified PCR product inhibits subsequent FokI digestion).

Step 2. FokI digestion. Purified DNA product of 0.1 ml PCR was incubatedwith FokI (New England Biolabs) at a ratio of >1.5 units per microgramof DNA (the DNA mass was estimated assuming that <100% of the primerswere converted into the full-length PCR product). This ratio had to beoptimized with every new DNA preparation. FokI digestion was carried outin 40:1 at 37° C. for 1 hour in the manufacturer's buffer with 0.1% ofTween-20 detergent to decrease the exonuclease activity.

Step 3. Klenow extension. dNTPs (final concentration of 0.5 mM each) andthe Klenow fragment of E. coli DNA polymerase 1 (from US Biochemicals,final concentration 370 units/ml) were added directly to the FokIdigest. Extension proceeded for 15 minutes at 37° C. The digested andblunt-ended library DNA was then purified from the other digestionfragments by native PAGE as in step 1. PAGE showed that 60% of the inputDNA was cut as expected, 40% was degraded nonspecifically, and anegligible fraction was left uncut.

Step 4. Ligation. The purified library DNA was resuspended in water andblunt-end ligated to the new fixed sequence. The new fixed sequence wasa duplex of two DNA oligonucleotides: C (5′-ggtgcggcagttcggt-3′ (SEQ IDNO:328)) and its complement, cC (5′-accgaactgccgcacct-3′ (SEQ IDNO:329)). The duplex was formed by incubation of the mixture of 200pmoles of each oligo in 4:1 of TE at 95° C. for 1 minute, followed by60° C. for 10 minutes and room temperature for 10 minutes. The duplexwas added to the purified DNA, and incubated with 2 units of T4 DNAligase (Roche Molecular Biochemicals) in the manufacturer's buffer in 20μl for 1 hour at 30° C. The ligation yield was 50%, estimated byMolecular Dynamics phosphorimager quantification of ³²P-labeled DNAseparated by PAGE. The overall yield of all steps was 10% relative tothe input DNA at the beginning of step 2. The relatively high blunt-endligation yield was achieved by keeping all DNAs as concentrated aspossible (more than a few :M), since the K_(m) of ligase for blunt endsis 50:M (Sugino el al. (1997) J. Biol. Chem. 252:3987-94). Theoligonucleotides, lacking a phosphate, cannot be ligated to anythingexcept the digested library DNA. To reduce ligation of the library DNAmolecules to each other, an excess of oligonucleotides over the libraryDNA was used (>2-fold excess, estimated by assuming that <100% of theprimers were converted into the full-length PCR product, and that <100%of it was recovered after gel purification). The length of the ligationproducts was verified by PAGE.

Step 5. PCR. One-third of the ligation product was amplified by PCR withthe new 3′ fixed sequence primer C (sequence shown above) and primerA+FokI (which introduces the FokI site into the old 5′ fixed sequence,and relates to primer A as B+FokI relates to B). Higher concentrationsof the primers were used in this PCR (10:M each, instead of 1:M, as inall other PCRs in this study). This served to provide an excess ofprimer C over cC (cC is complementary to C, and was carried over fromthe ligation in step 4).

Only one of the two major ligation products can be amplified in PCR withprimers C and A+FokI, namely, the product in which the duplex ofoligonucleotides C and cC has been ligated in one of the 2 possibleorientations with respect to the FokI-digested library DNA molecule. Asingle T was added to the 3′ end of oligonucleotide cC, as shown above,in order to create a single base overhang in the C-cC duplex. Under theexperimental conditions, adding a single overhanging T directs ligationmore toward the desired orientation: blunt end to blunt end, as opposedto the undesired orientation of overhanging end to blunt end (data notshown).

Step 6. Changing the 5′ fixed sequence. DNA was purified as in the thirdparagraph of step 1, and then steps 2-5 for 3′ fixed sequence wereessentially repeated for the 5′ fixed sequence. For the PCR in step 5,primers for the new fixed sequences were used: C (step 4, above) and D(5′-gaaattaatacgactcactatagggaaagcccacgcc-3′ (SEQ ID NO:330)). Theresulting molecules had both fixed sequences replaced with the new ones,with both tails removed entirely. One such molecule is shown in FIG. 2D.After changing the fixed sequences, SELEX proceeded as in “ConventionalSELEX.” with 1:M RNA and 100 nM MS2 CP. In the SELEX experiment wherethe new fixed sequences were chosen without the help of the STOGENcomputer program (see below), the primers that correspond to C and Dwere, respectively, 5′-atgtcgggccgccgaa-3′ (SEQ ID NO:331) and5′-gaaattaatacgactcactatagggcccggc gcataa-3′ (SEQ ID NO:332).

rffG End-Point Analysis

To find the sequences of the library molecules that share the rffG site,the method described previously (Singer et al., 1997, supra) was used.Briefly, the starting library DNA was amplified by PCR with the libraryprimer B, and the genomic primer from rffG gene(5′-bbbcactgagcatcagecag-3′ (SEQ ID NO:333); b stands for biotin). Theproducts that contained the genomic primer were purified on immobilizedstreptavidin. Their complementary strands were eluted, and amplifiedusing primer B and a nested genomic rffG primer(5′-bbbatcagccagactgtgtca-3′ (SEQ ID NO:334); the sequence in common isin boldface). The PCR products were purified on immobilized streptavidinagain, eluted, and amplified with uracil-primers for subsequent cloningand sequencing.

Binding Analysis

The MS2 replicase fragment (the natural MS2 CP binding site; FIG. 4) waschemically synthesized, amplified by PCR and labeled by in vitrotranscription as above. The resulting RNA molecule contained thefragment of the original MS2 sequence (as published in the GenBank) withthe same fixed sequences (for primers C and D) attached to it as in thereal SELEX isolates. The molecule also matched the SELEX isolates inlength (70 nucleotides, with most isolates being 60-80 nucleotides). TheMS2 CP binding site was positioned approximately in the middle of themolecule.

The RNAMOT site Nos. 8, 12 and 14 (Table 3) were amplified from E. coligenomic DNA template by PCR. The transcribed RNA molecules had the MS2CP binding sites positioned approximately in the middle Oust as theywere positioned for simplicity when their predicted secondary structureswere examined). The molecules also had the same fixed sequences, andwere 70 nucleotides long.

Labeled RNA (0.1 nM) was bound to MS2 CP in variable excessconcentrations as above, but without pre-filtering, at 24-25° C. Eachbinding reaction was filtered through nitrocellulose (0.45 micron pore,from Bio-Rad) using a Bio-Dot apparatus (Bio-Rad) and washed with 0.5 mlof the binding buffer.

STOGEN: A Computer Program to Choose New Fixed Sequences

To reduce the possible influence of the fixed sequences on the outcomeof the SELEX experiments, a computer program to design new fixedsequences was developed. The program (available by anonymous ftp from/usr/local/ftp/pub/STOGEN at beagle.colorado.edu or by e-mailing totimur@colorado.edu or javornik@nexstar.com) takes as input the old fixedsequences. It generates possible candidates for the new fixed sequences,using 4 heuristic rules with user-adjustable parameters (see below). Forcomputational efficiency, the program does not generate and test allpossible sequences of a given length, but rather randomly generates asubset of sequences, tests them, and repeats the process again, until itarrives at sequences that conform to all of the rules (hence the name ofthe program—STOGEN, short for stochastic generator). The STOGEN rulesare:

1. The new fixed sequences should have approximately the same annealingtemperatures to the primers as the old fixed sequences, in order tofacilitate amplification. Therefore, the new fixed sequences have thesame length and the same number of G+C as the old ones (Wu el al. (1991)Prog Nucleic Acid Res. Mol. Biol. 40:185-220).

2. The new fixed sequences should form among themselves as littlesecondary structure as possible. In addition to potentially influencingSELEX, the structure may hinder either PCR or reverse transcription. Thelongest allowed continuous stem was usually limited to 3 base pairs.

3. The new fixed sequences should share as little similarity as possibleto the old ones. Thus, all the molecules that used the old fixedsequences for binding, will be lost in subsequent rounds of SELEX. Twocriteria were used to this end: shared sequence size andposition-by-position identity. The maximum allowed size of any sequencein common between the old and the new fixed sequences was usually set to2 nucleotides. That is, if the old fixed sequence contains AUG, the newone may contain AU or UG, but not AUG. The only exception is theinvariant starting GGG, required for optimal transcription yield(Milligan et al. (1987) Nucleic Acids Res. 15:8783-98).

Also, position-by-position identity between the old and the new fixedsequences is limited. Since the parts of the fixed sequences closest tothe insert participate in binding more often, identity is weighted byposition, assigning greater penalty to positions closest to the insert.The weight function was derived by evaluating the fixed sequenceparticipation data from 11 different SELEX experiments. Sevenexperiments were randomized sequence SELEXes for protein binders (Brownet al. (1997) J. Biol. Chem. 272:14969-74; Brown and Gold (1995)Biochemistry 34:14765-74; Burke et al. (1996) J. Mol. Biol. 264:650-66;Jellinek et al. (1994) Biochemistry 33:10450-6; Kubik et al. (1994)Nucleic Acids Res. 22:2619-26; Tuerk et al. (1992) Proc. Natl. Acad.Sci. USA 89:6988-92; Tuerk & MacDougal-Waugh (1993) Gene 137(1):33-9),two were genomic SELEX experiments (experiments 1 and 2, FIG. 3)described in the present paper, one was SELEX for small-molecule binders(Burke et al. (1997) Chem. Biol. 4:833-43), and one was SELEX for RNAbest cleaved with an endonuclease (Jayasena et al. (1996) Biochemistry35:2349-56). In every SELEX experiment, the data generated for theindividual isolates by boundary experiments or by any other similarlyreliable method, were used. Although each SELEX experiment yielded verydifferent results, the cumulative data from all of these SELEXexperiments is approximated well by the formula:f=0.027+2.0×10⁻⁸×(n−20)⁶, for n≦10, andf=0.022, for n>10.

Here f is the relative frequency of participation of each positionwithin the fixed sequence, and n is the position (counting from theinsert, n=1 is the first fixed nucleotide). The program assigns theidentity score for each position to be f, if the new fixed sequence andthe old one both have the same nucleotide at this position, and to0—otherwise. New fixed sequences with a particularly high total identityscore (=the sum of the identity scores over all positions) are avoided.

4. The new fixed sequences should have minimal potential to formsecondary structure with the genomic insert. In most cases, the sequenceof the genomic insert is not known in advance. Hence, the generalizedpotential to form secondary structure is evaluated in the following way.

For each of the candidates for the new fixed sequences, the free energyof annealing to its complement is calculated (the complement used hereincludes A:U, G:C and G:U base pairs). For example, the sequence AC thushas one possible perfect complement, GU. The sequence GU has 4 possibleperfect complements: AC, AU, GC and GU, since U can base pair witheither A or G, and G—with either C or U.

The sum of free energies of all possible complements annealing to thefixed sequence is calculated using the Turner rules (Serra & Turner(1995) Methods Enzymol. 259:242-61). The more negative the sum, thelarger the potential to form secondary structure. Sequences with aparticularly negative sum of free energies (typically, G,U-rich) areavoided.

EXAMPLE 2

Binding Sites from the MS2 CP SELEX Agree with the Known ConsensusStructure

A library of genomic DNA was prepared from E. coli B by random primerextension (Singer et al. (1997) Nucleic Acids Research 25:781-786). Thelibrary contained approximately 65 nucleotide genomic inserts flanked byfixed sequences, which serve as primer annealing sites foramplification. Insert refers to the genomic sequence located in thelibrary molecule between the two fixed sequences. In each round ofSELEX, the transcribed library was allowed to bind MS2 CP, and then thebound RNA was amplified. In SELEX experiment 1, the DNA was cloned andsequenced after 5 rounds, when the optimal binding was observed.

Out of 25 isolates sequenced, 12 had the predicted consensus (Witherellet al. (1991) Prog Nucleic Acid Res. Mol. Biol. 40:185-220) binding site(FIG. 2A), which could be identified either by folding by hand, or bycomputerized Zuker-Turner folding (Genetics Computer Group, ProgramManual for the Wisconsin Package, 8^(th) edition, Madison, Wis., 1994;Serra and Turner (1995) Methods Enzymol. 259:242-61; Zuker (1 989)Science 244:48-52). Of the 12 isolates, 10 were found in the GenBank,which included the complete E. coli sequence, by the BLAST search(Altschul et al. (1990) J. Mol. Biol. 215:403-410) using the networkservice at the NCBI (Genetics Computer Group, Program Manual for theWisconsin Package, 8^(th) edition, Madison, Wis., 1994). The remaining 2isolates did not have significant similarities to any sequences inGenBank, and probably resulted from contamination of the startinggenomic library DNA.

Surprisingly, the genomic sequences, obtained from the GenBank, thatcorresponded to 9 out of the 10 isolates with the consensus bindingsite, did not contain this site. Thus, they were not predicted to bindMS2 CP. In other words, 9 out of the 10 isolates were experimentallyinduced artifacts.

One of the frequent artifacts is shown in FIGS. 2B and 2C. In thisisolate, the fixed sequence participates in forming the binding site.This isolate also has several mutations in the insert that participatein forming the binding site. All of the mutations are at the junctionwith the fixed sequence. This junction is much more prone to mutationsthan the rest of the genomic insert because of the random sequenceintroduced when the randomized primer misannealed during libraryconstruction (Singer et al. (1997) Nucleic Acids Research 25:781-786).The mutated region of the genomic insert at its junction with the fixedsequence is termed the tail.

In the isolate shown in FIGS. 2B and 2C, as well as in most otherisolates, the tail and the fixed sequence both participate in formingthe binding site. The corresponding genomic sequences from the GenBankwere very different from the tails and, obviously, from the fixedsequences, and thus were unable to form the binding site.

Note that the binding of the genomic sequences was not actuallymeasured, but rather inferred from the sequence. The sequence aloneshould predict the binding fairly well, as confirmed in this paper (seebelow), and as shown earlier by others (Schneider et al. (1992) J. Mol.Biol. 228:862-9; Stockley) et al. (1995) Nucleic Acids Res. 23:2512-8;Uhlenbeck et al. (1983) J. Biomol. Struct. Dyn. 1:539-52; Witherell etal. (1991) Prog Nucleic Acid Res. Mol. Biol. 40:185-220).

Annealing of the Complementary Oligonucleotides Reduces the Fraction ofSELEX Artifacts

It was desirable to reduce the fraction of isolates in which the fixedsequences, or tail, or both, participate in binding. The first method tosolve this problem was designed to reduce the participation in bindingof only the fixed sequences, but not the tails. The genomic SELEXdescribed in Example 1 above (termed conventional genomic SELEX) wascarried out for 3 rounds. In each subsequent round, two DNAoligonucleotides complementary to the two fixed sequences were annealedprior to binding of RNA to MS2 CP (Table 1). SELEX with annealing wascarried out for 3 rounds (FIG. 3, SELEX experiment 2). Switching from“no annealing” to “annealing,” rather than doing all 6 rounds“annealing.” should reduce the fraction of isolates that requireannealing for binding to MS2 CP.

Out of 35 sequenced isolates from “annealing” SELEX, 26 had theconsensus binding site, and 17 of those 26 were found in the GenBank. Ofthese 17 isolates, 7 (40%) had a consensus binding site present in thecorresponding genomic sequence from the GenBank, and the rest wereartifacts as described above. The fraction of artifacts in which fixedsequences, but not tails, participated in binding, decreased only byapproximately twofold.

Changing Fixed Sequences Eliminates Most of the SELEX Artifacts

The second, and more efficient, method of reducing the artifactsconsists of changing the fixed sequences sometime through the course ofSELEX, replacing them with entirely new fixed sequences, and at the sametime eliminating the “tails” altogether (FIG. 1).

FokI endonuclease was used to cut the 3′ fixed sequence and the tail ofthe library DNA after round 3 of conventional genomic SELEX. FokI cutsat a specific distance (9-13 nucleotides, regardless of their sequence)away from its recognition site, which was introduced in the fixedsequence near its junction with the genomic insert. After digestion withFokI, the overhang at the cut end of the library DNA was extended withKlenow, and blunt-end ligated to the new 3′ fixed sequence, which was aduplex of synthetic oligonucleotides. The ligation product was amplifiedby PCR, and the whole procedure was repeated again—this time to changethe 5′ fixed sequence. The new fixed sequences were chosen using aspecially developed computer program termed STOGEN (see Example 1). Thesequences may also be chosen manually, with the careful consideration ofall the important factors involved (discussed in Example 1).

After changing the 5′ and 3′ fixed sequences, SELEX was performed in 2different ways in parallel: (1) with annealing of the DNAoligonucleotides complementary to the new fixed sequences, and (2)without any complementary oligonucleotides, as in conventional genomicSELEX (FIG. 3.

SELEX experiments 3 and 4). Both SELEX experiments gave virtuallyidentical results. After 2 and 3 rounds of SELEX with the new fixedsequences, 101 isolates were sequenced. Out of 101 isolates, 76 had theconsensus binding site, and 75 out of these 76 were found in theGenBank.

The fixed sequences never made any part of the consensus binding site.Neither did the tails, except for 1 artifact. Practically all tails hadbeen successfully removed by FokI. Internal, rather than tail, mutationscaused 2 artifacts. This is comparable to the frequency of any mutationsin genomic SELEX (1.7 mutations per 100 nucleotides after 5-6 SELEXrounds, data not shown).

Five isolates closely resembled the consensus binding site, but hadmutations in those parts that do not contact the coat protein directly(Valegard et al. (1 994) Nature 3711:623-6; Valegard et al. (1997) J.Mol. Biol. 270:724-38). None of the F6-like, 3 nucleotide loop variants,which binds more weakly than the consensus binding site (Convery et al.(1998) Nat. Struct. Biol. 5:133-9), has been found in the genomic SELEXexperiments.

All of the SELEX isolates with the MS2 CP consensus binding site (Table2) had the higher-affinity RNCA tetraloop instead of the lower-affinityRNUA tetraloop of the natural MS2 CP binding site on MS2 mRNA (Johansson(1998) Proc. Natl. Acad. Sci. USA 95:9244-9; Lowary and Uhlenbeck (1987)Nucleic Acids Res. 15:10483-93). As expected, these SELEX isolates boundbetter than the natural binding site, as measured by the nitrocellulosefilter-binding assay (FIG. 4). Four most frequent isolates without theconsensus binding site were also tested. Three isolates did not bind MS2CP (a typical one is shown in FIG. 4), and one bound nitrocellulose andalso, weakly, MS2 CP (data not shown).

The consensus of the isolates in Table 2, with consideration of theirfrequencies in the selected pool, is shown in FIG. 2 e. Most of thedifferences between the SELEX consensus site and the consensus site inFIG. 2A, should make the binding tighter, or the binding structure morestable (Uhlenbeck et al. (1983) J. Biomol. Struct. Dyn. 2:539-52;Witherell et al. (1991) Prog Nucleic Acid Res. Mol. Biol. 40:185-220).This SELEX consensus also agrees with the data from the randomizedsequence SELEX (Schneider et al. (1992) J. Mol. Biol. 228:862-9).

The isolates that bind MS2 CP were located in 8 distinct genomic sites.Locations of the MS2 CP binding sites within the corresponding genes didnot follow any obvious pattern, with some sites being on the sense, andsome on the antisense strand.

Fifty-six isolates were from the sense strand of mRNA of the rffG gene(FIG. 2D). These molecules were not only the most frequent, but also thetightest binders among the SELEX isolates. The corresponding genomicfragment (without the fixed sequences) also bound MS2 CP well (FIG. 4).The rffG open reading frame, o355, was mistakenly labeled rffE in theGenBank version 90.0 (Marolda and Valvano (1995) J. Bacteriol.177:5539-46). Encoded by rffG is the enzyme dTDP-D-glucose 4,6dehydratase. This enzyme participates in formation of O-specificpolysaccharide, or O antigen, which, joined together with lipid A viacore oligosaccharide, forms lipopolysaccharide in the bacterial outermembrane (Raetz, Escherichia Coli and Salmonella: Cellular and MolecularBiology. 2^(nd) edition, 1996, pp. 1035-1063). The enzyme alsoparticipates in formation of the polysaccharide part of theenterobacterial common antigen, a cell surface glycolipid (Rick &Silver, Escherichia Coli and Salmonella: Cellular and Molecular Biology,2^(nd) edition, 1996, pp. 104-122).

Library Composition Partially Explains SELEX Results

It is worth noting that 93% of all the rffG isolates were virtuallycopies of each other, with the 15 nucleotide consensus binding sitealways starting at any of only 4 adjacent positions (or adjacent“registers”) within the 40 nucleotide insert. The site was predictedideally to shift everywhere within the 40−15+1=26 available registerswithin the insert. The fact that it did not, was possibly due to a biasin the starting library. The end-point analysis (Singer et al. (1997)Nucleic Acids Research 25:781-786) showed that more than half of themolecules of the starting library have end-points that correspond to theregisters of the majority (93%) of all rffG SELEX isolates (FIG. 5A).Such clustering of end-points might have been caused by annealing of thefixed part of the library primer to the E. coli DNA during the libraryconstruction (FIG. 5B).

Comparison of the MS2 CP Binding Sites Predicted by the Computer Searchwith the Sites Found by SELEX

Other sites in the E. coli genome that bind to MS2 CP were much lessfrequent than rffG, in these (Table 2) and prior rounds of SELEX (datanot shown). This raised the question about the efficiency of finding allpotentially biologically important binding sites. To check if any otherbinding sites were missed by the SELEX process, a search was performedfor the MS2 CP consensus binding site (FIG. 2A) in the complete E. coligenome, using the RNAMOT program (Gautheret et al. (1 990) Comput. Appl.Biosci. 6:325-31). The search revealed 412 matches to the consensusbinding site, each of which theoretically binds the coat protein as wellas, or better than, the wild-type MS2 mRNA. Only 280 sites were expectedby chance.

To narrow this list to only the tightest binding sites, the SELEXconsensus binding site (FIG. 2E) was searched for. It is based on thegenomic SELEX isolates, and is more restrictive than the consensusbinding site, which is based on the studies of mutants. RNAMOT found 21such “SELEX consensus” binding sites (Table 3). Only 3 sites wereexpected to be found at random.

Three binding sites were found both by SELEX and by RNAMOT program,including the major (rffG) SELEX isolate. Most of the minor SELEXisolates were not found by RNAMOT. Some of these did not fit the SELEXconsensus used for searching the database. For example, had G:U pairsbeen allowed in the consensus, these sites would have been found, too.Others, like isolate Nos. 7 and 9 from Table 2, were not in thedatabase.

Most of the RNAMOT sites were not found by the SELEX process. It ispossible that some of them were under-represented in the startinglibrary, or that they were poorly amplifiable in the SELEX process, orthat they bound MS2 CP weakly because the RNA folds into alternate,non-binding, structures within the context of a larger molecule. It isunlikely that these sites were entirely absent from the library. In aprevious experiment, all of the 13 other independently tested genomicfragments were successfully amplified by PCR from the same E. coligenomic library (Singer et al. (1997) Nucleic Acids Research25:781-786).

To find out why the RNAMOT sites were not isolated in SELEX, 20-40 moststable predicted secondary structures of some RNAMOT sites were comparedto those of the actual SELEX isolates. In addition, structures of therffG binding site present in all possible registers within the insertwere examined. There appeared only a weak correlation between structuresand the frequency of isolation in SELEX. The absolute free energy of thebinding site structure, or of the whole molecule, was not correlatedwith its isolation in SELEX. However, the major rffG isolate had afairly long stem that supported the binding site (FIG. 2D), relative toother SELEX isolates, and to many other rffG registers.

Perhaps a longer stem provides extra stability to the correct bindingsite structure and thus reduces the fraction of molecules folded intoother, non-binding, structures. In the randomized sequence SELEX for R17coat protein binders, Schneider and coworkers also found mostly long (7base pairs) and stable (mostly G:C or C:G base pairs) stems (Schneideret al. (1992) J. Mol. Biol. 228:862-9). Also, in the regular MS2 CPgenomic SELEX experiment (without changing the fixed sequences orannealing of oligonucleotides; FIG. 3, experiment 1), many isolates usedfixed sequences not only to form the consensus binding site, but also toextend its stem past the minimum 5 base pairs. In SELEX experiments 3and 4, the fixed sequences, while not forming the consensus bindingsite, sometimes extended the stem to longer than the minimal 5 basepairs. In SELEX experiment 5, the new fixed sequences were chosenwithout STOGEN, and their potential to base-pair to each other and tothe insert was accidentally overlooked. In most of the isolates fromthis SELEX experiment, the fixed sequences extended the minimal stem byadditional 12 base pairs.

However, when RNAMOT sites were considered as well, longer stems did notcorrelate with the frequency of isolation in SELEX. For example, siteNos. 8, 12 and 14 (Table 3), which were found by RNAMOT, are alsopredicted to have long stems, just like the most frequent genomic SELEXisolates (Nos. 1, 2 and 3, Table 2), and yet they were not found in theSELEX process. They fit the randomized sequence SELEX consensus stems(Schneider et al. (1992) J. Mol. Biol. 228:862-9) as well or better thanthe most frequent genomic SELEX isolates. They also bind MS2 CP ratherwell. Site Nos. 8 and 14 bind to MS2 CP with affinities between those ofSELEX isolate Nos. 1 and 2 (data not shown). Site No. 12 binds MS2 CPwith affinity only slightly weaker than SELEX isolate No. 6.

In short, the only parameters that apparently affect isolation of anyparticular molecule in the SELEX process are the molecule's affinity andits frequency in the starting library. Perhaps the sites predicted byRNAMOT, but not found in SELEX, were less frequent in the library.

Examples 1 and 2 demonstrate methods for generating nucleic acid ligandsin which the participation of the fixed region in binding to the targetis minimized or eliminated by changing fixed region sequences or byannealing of complementary nucleotides to the fixed regions. Thesemethods were demonstrated using the Genomic SELEX methodology; however,it would be known by one of skill in the art that these methods are notspecific to the Genomic SELEX methodology, but can by applied to themore broad SELEX methodology.

EXAMPLE 3

This Example provides general procedures followed by and incorporatedinto Examples 4-12.

Materials

Recombinant human Transforming Growth Factor Beta 1 (hTGFβ1) and humanVascular Endothelial Growth Factor (VEGF) were from R&D Systems(Minneapolis, Minn.). DNA and RNA modifying enzymes were from RocheMolecular Biochemicals (Indianapolis, Ind.), BRL (Gaithersburg, Md.), orNEB (Beverly, Mass.) or PE (Foster City. Calif.). Biotin-21-dUTP wasfrom Clontech (Palo Alto, Calif.), terminal deoxynucleotidyl transferasewas from Clontech (Palo Alto, Calif.) or Roche Molecular Biochemicals(Indianapolis, Ind.). T7 RNA polymerase, 2′F-modified CTP and UTP wereprepared in house. Taq DNA polymerase was from Perkin Elmer (FosterCity, Calif.). DNA oligonucleotides were obtained from OperonTechnologies, Inc. (Alameda, Calif.). All other reagents and chemicalswere from commercial sources.

Affinity Selection (SELEX)

The SELEX procedure has been described in detail in the SELEX PatentApplications. The DNA templates contained either 40 or 30 randomnucleotides, flanked by 5′ and 3′ constant regions for primer annealingsites for PCR and cDNA synthesis (Table 5). Truncation SELEX startedwith VEGF round 12 VT30 or TGF∃1 round 13 40N7 or 30N7 pools. Thesepools were described previously (see Ruckman et al. (1998) J. Biol.Chem. 273:20556-67 and U.S. Ser. No. 09/046,247, filed Mar. 23, 1998).Selection conditions for truncation SELEX are summarized in Tables 6A-D.RNA pools were prepared by transcription with about 5 μM DNA template, 5units/μl T7 RNA polymerase, 40 mM Tris-HCl (pH8), 12 mM MgCl₂, 5 mM DTT,1 mM spermidine, 0.002% Triton X-100, 4% PEG 8000, 2-4 mM each 2′OH ATP,2′OH GTP, 2′F CTP, 2′F UTP, and 0.25 μM α³²P-ATP (800 Ci/mmole). Whennecessary, RNA pools were prefiltered and/or preadsorbed with multiplelayers of same nitrocellulose filter type used in the SELEX process inorder to reduce the frequency of molecules selected for nitrocellulosebinding. To prepare binding reactions, the RNA molecules were incubatedwith recombinant h TGF∃1 in Dulbecco's Phosphate-Buffered Saline (DPBS)(Life Technologies, Gaithersburg, Md.) containing 1 mM MgCl₂ and 0.01%human serum albumin or with recombinant VEGF in TBS (Sambrook et al.,Molecular Cloning: A Laboratory, Manual, 2^(nd) Edition, Cold SpringHarbor, N.Y., 1989) containing 1 mM MgCl₂, 1 mM CaCl₂ and 0.01% humanserum albumin. Following incubation at 37° C. (about 30 minutes) theprotein-RNA complexes were partitioned from unbound RNA by capture onnitrocellulose. Nitrocellulose filter bound RNA was recovered byphenol/urea extraction. Pools were amplified by RT/PCR or PCR. Reversetranscriptions were done by AMV reverse transcriptase at 48° C. for 60minutes in 50 mM Tris-HCl pH 8.3, 60 mM NaCl, 6 mM Mg(OAc)_(2, 10) mMDTT, 50 pmol DNA 3′ primer (3G7) (Table 5), 0.4 mM each of dATP, dCTP,dGTP, and dTTP, and 1 unit/μl AMV RT. PCR amplifications were done with2 μM each 3G7 and 5G7 primers (FIG. 1), 50 mM KCl, 10 mM Tris-HCl, pH 9,0.1% Triton X-100, 3 mM MgCl₂, 0.5 mM of each dATP, dCTP, dGTP, anddTTP, 0.1 units/μl Taq DNA polymerase. Typically 15 cycles were used of30 seconds denaturation at 95° C., 30 seconds annealing at 60° C., and 1minute elongation at 72° C.

Nitrocellulose Filter Partitioning

To partition the protein-RNA complexes away from uncomplexed RNA, thebinding reactions were filtered through nitrocellulose/celluloseacetated mixed matrix, 0.45 μm pore size filter disks, type HA,(Millipore, Co., Bedford, Mass.). For filtration, the filters wereplaced onto a vacuum manifold and wetted by aspirating 5 ml of bindingbuffer. The binding reactions were aspirated through the filters, thenfilters were washed with 5-50 ml of binding buffer (without BSA) andcounted in a scintillation counter (Beckmann). When necessary,nitrocellulose filters were preblocked with 2 ml of PBS +0.01% BSA toreduce background binding of RNA.

Nitrocellulose partitioning was also used for determining theequilibrium dissociation constants of RNA ligands to hVEGF or hTGFβ1.High specific activity transcripts for affinity determination wereprepared in 20 μl reactions containing 5 μl crude PCR product and 15 μltranscription mix (3.9 μl 5× transcription buffer, 0.1 μl 1 mM ATP, 0.2μl 100 mM GTP, 0.6 μl 100 mM 2′F-CTP, 0.6 μl 100 mM 2′F-UTP. 2.5 μlα-³²P-ATP (800 Ci/mmol, NEN, Boston, Mass.), 0.5 μl T7 RNA polymerase(at 14.7 μM)). Following incubation (1 hour—overnight), transcripts weregel purified from denaturing polyacrylamide TBE gels (for example 10% or15%, Novex, San Diego, Calif.) and were used at about 25,000-50,000 cpmper 8-12 point binding curve. Binding curves obtained by nitrocellulosefiltration indicated that RNA pools and some RNA ligands bindmonophasically while others bind biphasically. Biphasic binding can bedescribed as the binding of two affinity species derived from the sameligand sequence that can fold into alternate structures that arekinetically trapped and are not in equilibrium.

To obtain the monophasic equilibrium dissociation constants of RNAligands to hTGFβ1the binding reaction:KDR:P→R+P

-   -   R=RNA    -   P=Protein    -   K_(D)=dissociation constant        is converted into an equation for the fraction of RNA bound at        equilibrium:        q=(f/2R _(T))(P _(T) +R _(T) +K _(D)−((P _(T) +R _(T) +K        _(D)))²−4P _(T) R _(T))_(1/2))    -   q=fraction of RNA bound    -   P_(T)=total protein concentration    -   R_(T)=total RNA concentration    -   f=retention efficiency of RNA-protein complexes        The average retention efficiency for RNA-TGF∃1 complexes on        nitrocellulose filters is 0.4-0.8. Biphasic binding data were        evaluated with the equation:        q=2P_(T) +R _(T) +K _(D1) +K _(D2)−[(P _(T) +X ₁ R ₁ +K        _(D1))₂−4P _(T) X ₁ R _(T)]^(1/2)−[(P ^(T) +X ₂ R _(T) +K        _(D2))²−4P _(T)X₂R_(T)]^(1/2),        where X₁ and X₂ are the mole fractions of the affinity species        R₁ and R₂ and K_(D1) and K_(D2) are the corresponding        dissociation constants.

The K_(D)s were determined by least square fitting of the data pointsusing the software Kaleidagraph (Synergy Software, Reading, Pa.).

RNaseH Digestion

Primer binding sites of RNA transcripts were removed by RNaseH digestionas follows. Gel purified 2′fluoro-pyrimidine modified RNA (about 200pmoles) was incubated with 0.1 mM each final concentration of 3H7 and5H7 oligonucleotides (Table 7) in 50 μl volume. Thetranscript-oligonucleotide mix was heated at 95° C. for 4 minutes,incubated at 50° C. for 10 minutes and at 37° C. for 2 minutes and wassupplemented with 5 μl of 10× RNaseH buffer (200 mM Hepes-KOH, pH 9.0,500 mM KCl, 100 mM MgCl₂) and 25 μl of RNaseH (1 U/μl, Roche MolecularBiochemicals, Indianapolis, Ind.). The transcripts were digested at 37°C. for 30-60 minutes and then were ethanol precipitated in 2 M ammoniumacetate in the presence of 20 μg of glycogen carrier (Roche MolecularBiochemicals, Indianapolis, Ind.) and resuspended in appropriate volumeof H₂O. RNaseH digestion of small amounts (0.03-0.5 pmoles) of highspecific activity RNA was done in a smaller volume (usually 20 μl) inthe presence of 1 unit of RNaseH and 250 nM each 3H7 and 5H7.

Mapping of RNaseH Digestion Sites

To map RNaseH digestion sites, 2′F RNA transcripts of ligands VT30-07and VT30-44 (Ruckman et al. (1998) J. Biol. Chem. 273:20556-67) were ³²Plabeled at their 5′ or 3′ ends. For 5′ end labeling, 2.5 pmols of eachtranscript were phosphatased in 25 μl reactions containing 2.5 unitsshrimp alkaline phosphatase (Roche Molecular Biochemicals cat#1 758250)in manufacturer's specified buffer, for 60 minutes at 37° C. Followingheat inactivation at 70° C. for 20 minutes, the phosphatased RNA waskinased with T4 polynucleotide kinase (Roche Molecular Biochemicals cat#709557) per manufacturer's instruction and 2 μCi γ³²P-ATP (10 Ci/mmole,New England Nuclear, cat#NEG502) at 37° C. for 30 minutes. For 3′ endlabeling, 10 pmols of each transcript were incubated with T4 RNA ligase(Roche Molecular Biochemicals cat#1449478) and 10 pmols of Cytidine3′,5′-bis(phosphate), [5′-³²P] (3000 Ci/mmol, New England Nuclear, cat#NEG019A) in 30 μl (total) of reaction buffer containing 10% DMSO.Ligation reactions were incubated at 4° C. overnight. Labeled RNA wasrecovered by ethanol precipitation in the presence of glycogen ascarrier (Roche Molecular Biochemicals, cat# 901393) and gel purifiedfrom a denaturing 10% polyacrylamide 8M urea gel. About 10⁵ cpmequivalents of labeled RNA was then digested by RNaseH as describedbefore using such oligonucleotides to digest the 5′ end labeled RNA atits 3′ primer binding site while the 3′ end labeled RNA at its 5′ primerbinding site. Following RNaseH digestion, the RNAs were recovered byethanol precipitation with glycogen as above. To prepare size markers,RNA equivalent to 2.5×10⁵ cpm were subject to alkaline hydrolysis asfollows. RNAs were incubated in 50 mM NaCO₃ pH 9.75 at 92° C. for 12minutes and was then neutralized with 2 μl (a tenth volume) 3MNa(CH₃COO) pH 5.5. Hydrolyzed RNAs were recovered by ethanolprecipitation as above. RNaseH digested and alkaline hydrolyzed RNAswere then analyzed on a 15% acrylamide, 8M urea gel.

Mapping of 5′ RNaseH Digestion Sites by Primer Extension

To map the RNaseH digestion sites by primer extension, 200 pmol 2′F RNAtranscripts of ligands VT30-07 and VT30-44 (Ruckman et al. (1998) J.Biol. Chem. 273:20556-67) were RNaseH digested and gel purified asabove. Primer 3G7 was 5′ end labeled with T4 polynucleotide kinase(Roche Molecular Biochemicals cat# 709557) per manufacturer'sinstructions and 4 μCi γ³²P-ATP (10 Ci/mmole, New England Nuclear,cat#NEG502) at 37° C. for 30 minutes followed by heat inactivation at70° C. for 10 minutes. For sequencing reactions, 0.5 pmoles of template(RNaseH digested VT30-01 or VT30-44) were mixed with 5 pmoles of kinased3G7 primer in 10 μl 1×RT-no-Mg buffer (50 mM Tris-HCl, pH 8.6, 60 mMNaCl, 10 mM DTT), incubated at 70° C. for 5 minutes and chilled on ice.Extension reactions were set in 5 μl volumes, utilizing 2 μl of theannealing mix, and 0.2 mM (final concentration) dideoxy mix (A or C or Gor T) in 1× RT buffer (50 mM Tris-HCl pH 8.6. 60 mM NaCl, 10 mM DTT, 6mM Mg(CH₃COO)₂) and 5 U AMV RT (Boheringer Mannhein cat# 1495062).Dideoxy mixes were prepared in 5× concentration containing one dideoxynucleotide at 1 mM and each dNTP at 1.87 mM, in 1× RT buffer (50 mMTris-HCl, pH 8.6, 60 mM NaCl, 10 mM DTT, 6 mM Mg(CH₃COO)₂) supplementedto 24 mM Mg(CH₃COO)₂). Extension reactions were incubated at 37° C. for15 minutes, supplemented with formamide dye, denatured at 95° C. forfive minutes and analyzed on an 8% polyacrylamide, 8 M urea gel.

Short Primer RT/PCR

Primers and templates are as shown in Table 8. RT reactions were set in50 μl volume 1× reaction buffer (manufacturer supplied), containing 1 μMprimer 3GTR, 0.4 mM each dNTP and 25 Us AMV RT (Boheringer Mannhein cat#1495062). Prior to adding the RT, reaction mixes were denatured at 95°C. for 3 minutes, annealed at 40° C. for 10 minutes and following theaddition of RT, they were incubated at 40° C. for 45 minutes. RTreactions were then supplemented to 100 μl PCR reactions containing 1×buffer (manufacturer supplied) 3 mM MgCl₂. 0.5 mM each dNTP, 1 μM each3GTR and 5GTR primers, 10% DMSO and, 5 Us Taq DNA polymerase (PerkinElmer, cat#1248). PCR reactions were denatured at 93° C. for 3 minutesand then cycled (20-25 cycles) at 93° C. 2 minutes, 40° C. 1 minute. 72°C. 1 minute. PCR products were analyzed on 10% polyacrylamide TBE gelsand used for transcription without purification.

RNA Ligation

Primer binding sites at the 3′ end were introduced to RNA using RNAligations by mixing 16 μl (about 1 pmole) RNA, 3 μl 10× buffer (500 mMHEPES pH 7.8, 200 mM MgCl₂, 35 mM DTT, 100 μg/ml BSA), 1 μl 3′ oligo(5′-CAGACGACTCGCCCGA (SEQ ID NO: 174) or 5′-GACGACTCGCCCGA (SEQ IDNO:175)) at 20 μM, 6 μl DMSO, and 1 μl RNA ligase (10 U/μl, RocheMolecular Biochemicals, Indianapolis, Ind.). Reactions were incubated at4° C. for 12-16 hours and ligated RNA was purified by ethanolprecipitation in the presence of 10 μg glycogen. Purified RNA usuallywas resuspended in water.

DNA Ligation

Primer binding sites at the 5′ end were introduced by ligation at the 3′end of cDNA synthesized on selected RNA. For reverse transcription, to18 μl of 3′ end ligated RNA we added 2 μl of 3G7 primer (Table 5) at 100μM, and the mix was annealed by heating at 95° C. for 5 minutes, then at55° C. for 10 minutes, followed at 37° C. for 15 minutes and 25° C. for5 minutes. Following annealing, the reaction was supplemented with 8 μl5× superscript builder (Roche Molecular Biochemicals, Indianapolis,Ind.), 5 μl H₂O, 4 μl DTT at 100 mM, 2 μl dNTPs at 10 mM each, and 1 μlsuperscript (200 U/μl, Roche Molecular Biochemicals, Indianapolis,Ind.). The reaction was then incubated at 45° C. for 45 minutes, theenzyme was heat inactivated at 95° C. for 5 minutes, and the cDNA wasethanol precipitated in 2M ammonium acetate, resuspended in 14 μl waterand carried into a ligation reaction where it was supplemented with 2 μlpreannealed bridge/linker (Table 4. Set One or Two) at 10 μM, heated at42° C. for 10 minutes and 25° C. for 10 minutes, supplemented with 2 μlligase storage buffer (Roche Molecular Biochemicals, Indianapolis,Ind.), 2 μl 10× ligation buffer (Roche Molecular Biochemicals,Indianapolis, Ind.),and 1 μl of T4 DNA ligase (5 U/μl, Roche MolecularBiochemicals, Indianapolis, Ind.), and incubated at 16° C. overnight.Half of the ligated cDNA was PCR amplified as follows. In PCR tubes wemixed 14 μl H₂O, 2.5 μl 10× amplitaq buffer (Perkin Elmer, Foster City,Calif.), 2.5 μl MgCl₂ at 25 mM, 2 μl each 5′(5′N7) and 3′ (3G7) primer(Table 4 and Table 5, respectively), and 2 μl of dNTPs at 10 mM each.The mix was then sealed with wax (ampliwax, Perkin Elmer. Foster City,Calif.), and supplemented above the wax layer with 53 μl H₂O, 7.5 μl 10×amplitaq buffer, 3.5 μl MgCl₂ at 25 mM, 10 μl ligated cDNA, and 1 μl Taqpolymerase (Perkin Elmer, Foster City, Calif.). The mix was cycled 25times at 54° C. (annealing) for 30 seconds, 72° C. (extension) for 1minute, and 95° C. (denaturation) for 30 seconds. Amplified productswere ethanol precipitated and gel purified on a 6% polyacrylamide TBEgel and reamplified for an additional 10 cycles as above. Finalamplified DNA was used as template for transcription.

Truncation SELEX by Ligation

SELEX pools were in vitro transcribed and the generated RNA was digestedwith RNaseH to remove either both (VEGF) or just the 5′ primer bindingsite (TGFβ1). Digested RNA was reverse 20 transcribed following (ifappropriate) ligation of 3′ primer binding site at its 3′ end. GeneratedcDNA was ligated with preannealed oligonucleotides 5N7 Linker (5′pCTCCCTATAGTGAGTCGTATTA (SEQ ID NO:36)), and 5N7 bridge(TAATACGACTCACTATAGGGAGNNNNN (SEQ ID NO:37)) as described above (Table4). These oligos introduce the T7 promoter and a transcriptioninitiation site GGGAG at the 5′ end of the library's sense strand whileremoving, the bulk of the 5′ primer binding site (5′Trunc-Library).Resulting cDNA is PCR amplified to generate the starting library asdescribed above. For affinity selection, the 5′Trunc-Library istranscribed with about 5 μM DNA template, 5 units/μl T7 RNA polymerase,40 mM Tris-HCl (pH 8), 12 mM MgCl₂, 5 mM DTT, 1 mM spermidine, 0.002%Triton X-100, 4% PEG 8000, 2-4 mM each 2′OH ATP, 2′OH GTP, 2′F CTP, 2′FUTP, and 0.25 μM α³²P-ATP (800 Ci/mnmole). The 3′ primer binding site isthen removed by RNaseH digestion. Resulting truncate RNA is thenincubated with recombinant h TGF∃1 in Dulbecco's Phosphate-BufferedSaline (DPBS) (Life Technologies, Gaithersburg, Md.) containing 1 mMMgCl₂ and 0.01% human serum albumin or with recombinant VEGF in TBS(Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd)Edition, Cold Spring Harbor, N.Y., 1989) containing 1 mM MgCl₂, 1 mMCaCl₂ and 0.01% human serum albumin. Following incubation at 37° C.(about 30 minutes) the protein-RNA complexes were partitioned fromunbound RNA by capture on nitrocellulose. Nitrocellulose filter boundRNA was recovered by phenol/urea extraction. When necessary, RNA poolswere prefiltered and/or preadsorbed with multiple layers of samenitrocellulose filter type used in the SELEX process in order to reducethe frequency of molecules selected for nitrocellulose binding. Affinityselected RNA was ligated to the 3′primer binding site, and was reversetranscribed into cDNA as described above. Resulting cDNA was ligatedwith preannealed oligonucleotides 5G7 RC Linker (5′pTATAGTGAGTCGTATTA(SEQ ID NO:34)) (Table 4), and 5′N7 (TAATACGACTCACTATAGGGAG (SEQ IDNO:46)) (Table 8) and PCR amplified as described above to generate thenext Truncate SELEX pool.

Streptavidin Gel Shift on Denaturing Gels

Streptavidin gel shifts on denaturing gels (Pagratis (1996) NucleicAcids Research 24:3645-6) was used to either analyze the presence ofbiotin on oligonucleotides or to purify, ssDNA following PCR. Briefly,biotinylated nucleic acid was mixed with 0.5 mg/mil (final) streptavidin(Pierce, Rockford, Ill.) in buffer (PBS, TFBS, or PCR), incubated atroom temperature for 10 min and mixed with equal volume of 2× formamidedye (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2_(nd)Edition, Cold Spring Harbor, N.Y., 1989). Following denaturation at 95°C. for 5 minutes, the mix was resolved on a 10% polyacrylamide 7Mdenaturing TBE gel (Novex, San Diego, Calif.). To purify end-labeledssDNA template strand from SELEX pools, 3′ primer was first end labeledby mixing 1 μl of primer at 1 mM, 2 μl of 10× T4 polynucleotide kinase(PNK) buffer (Boheringer Mannhein, Indianapolis, Ind.), 1 μl PNK(Boheringer Mannhein, Indianapolis, Ind.) at 10 u/μl, and 16 μl ofγ-³²P-ATP (3000 Ci/mmole, NEN, Boston, Mass.), and incubating at 37° C.for 30 minutes. The labeled primer (in 20 μl) was then heat treated at65° C. for 10 minutes and mixed with 5 μl crude PCR template (SELEXpool), 40 μl 10× PCR buffer (Perkin Elmer, Foster City, Calif.), 48 μl25 mM MgCl₂, 20 μl 10 mM each dNTP, 1 μl 1 mM 5′ primer which contains 3biotin molecules at its 5′end (incorporated as phosphoramidites, OperonTechnologies, Alameda, Calif.), and 4 μl Taq polymerase (Perkin Elmer,Foster City, Calif.), in 400 μl final volume and was PCR amplified for15 cycles as described before. The volume of the amplified DNA was thenreduced to 50 μl using microcon 30 cartridges (Amicon, Beverly, Mass.),mixed with 5 μl of streptavidin (Pierce, Rockford, Ill.) at 5 mg/ml,incubated at room temperature for 10 minutes, mixed with equal volume of2× formamide dye, and denatured and electrophoresed as described above.Following electrophoresis the radioactive band was purified bycrushing-soaking and ethanol precipitation.

RNA 3′ Biotinylation Using RNA Ligase

Acceptor RNA (0.1-10 pmole) was incubated with 20-200-fold excess donordinucleotide (FIG. 15), 1 mM ATP, 20% DMSO, and 50 units of T4-RNAligase (BMB) in 30 μl of 1× reaction buffer (50 mM HEPES pH 7.8, 3.5 mMDTT, 20 mM MgCl₂, 10 μg/ml BSA) for 16 hours at 4° C. Labeled RNA wasused as is or after ethanol precipitation.

RNA 3′ Biotinylation Using Terminal Deoxynucleotidyl Transferase

Selected RNA was biotinylated by terminal deoxynucleotidyl transferaseby mixing 3 μl RNA (about 1 pmole), 2 μl 5× (Clontech, Palo Alto,Calif.) buffer, 0.5 μl 25 mM CoCl₂, 2 μl 0.5 mM Biotin-21-dUTP(Clontech, Palo Alto, Calif. or Roche Molecular Biochemicals,Indianapolis. Ind.), and 2 μl terminal deoxynucleotidyl transferase (25U/μl, Clontech, Palo Alto, Calif. or Roche Molecular Biochemicals,Indianapolis, Ind.), in total of 10 μl reaction volume. Reactions wereincubated at 37° C. for 3 hours and labeled RNA was used as is or alterethanol precipitation.

Hybrid Formation

Selected RNA was biotinylated and then was mixed with ssDNA templatedstrands purified (as described) at 10× excess DNA over RNA, 1000× excesssynthetic 40-mer oligonucleotide DNA randomized at each position, 2 μl10× renature buffer (100 mM Tris, pH 7.5, 10 mM EDTA), 2 μl 500 mM NaClin a total volume of 18 μl. The mix was heated at 95° C. for 4 minutes,transferred on ice for 10 minutes, supplemented with 2 μl of 10 mMcetyltrimethylammonium bromide (CTAB), and annealed overnight at 72° C.Following overnight hybridization the reaction was stopped by adding 30μl of 1× renature buffer containing 0.2% SDS, and ethanol precipitatedin 2 M ammonium acetate and 10 μg glycogen (Roche MolecularBiochemicals, Indianapolis, Ind.). The pellet was resuspended in 1×renature buffer and hybridized ssDNA strands were capture bystreptavidin gel shift on native gels or by streptavidin agarose beads(cat# 20349, Immunopure immobilized streptavidin, Pierce, Rockford,Ill.).

Hybrid Selection by Streptavidin Beads

Following hybridization and ethanol precipitation, the reactions wereresuspended in 100 μl 1× renature buffer-50 mM NaCl.Streptavidin-agarose beads (50 μl) were washed in 0.45 μm spin-X filterunits (costar, Cambridge, Mass.), 2 times with 250 μl 1× renaturebuffer-50 mM NaCl and incubated with the 100 μl of hybridization mix for10 minutes at room temperature. Noncaptured nucleic acid was removed andthe beads were washed 3 times with 250 μl 1× renature buffer-50 mM NaCl(or until counts are no longer in the wash) and captured ssDNA waseluted by adding 100 μl H₂O incubated at 95° C. for 5 minutes. CollectedssDNA was supplemented with 20 μl 10× Taq buffer, 24 μl 25 mM MgCl₂, 10μl 10 mM each dNTPs,0.5 μl each 1 mM 3G7 and 5G7, 2 μl Taq polymerase intotal volume of 200 μl and PCR amplified taking 30 μl samples after 3,6, 9, 12, and 15 cycles for electrophoretic analysis (8 μl). The PCRaliquot with the best signal to noise ratio was amplified further in 400μl PCR reactions for additional 15 cycles.

Hybrid Selection by Gel Shift

Following hybridization and ethanol precipitation, the reactions(usually 15 μl total) were supplemented with streptavidin at 2.5 mg/ml(final concentration), incubated at room temperature for 10 minutessupplemented with 6× glycerol dye to final 1× and electrophoresed on a6%, 0.5× TBE gel, at 150 volts, for 30 minutes at room temperature.Streptavidin shifted bands were visualized following auto-radiographyand excised to recover ssDNA following crush-soaking and ethanolprecipitation. Eluted ssDNA was then PCR amplified as in the hybridselection by streptavidin beads.

Truncation SELEX by Hybrid Selection

SELEX pools were in vitro transcribed and the generated RNA was digestedwith RNaseH to remove both primer binding sites. Resulting truncate RNAwas then incubated with recombinant h TGF∃1 in Dulbecco'sPhosphate-Buffered Saline (DPBS) (Life Technologies, Gaithersburg, Md.)containing 1 mM MgCl₂ and 0.01% human serum albumin or with recombinantVEGF in TBS (Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) Edition, Cold Spring Harbor, N.Y., 1989) containing 1 mM MgCl₂, 1mM CaCl₂ and 0.01% human serum albumin. Following incubation at 37° C.(about 30 minutes) the protein-RNA complexes were partitioned fromunbound RNA by capture on nitrocellulose. Nitrocellulose filter boundRNA was recovered by phenol/urea extraction. When necessary. RNA poolswere prefiltered and/or preadsorbed with multiple layers of samenitrocellulose filter type used in SELEX in order to reduce thefrequency of molecules selected for nitrocellulose binding. Affinityselected RNA was biotinylated with either terminal deoxynucleotidyltransferase or RNA ligase as described above, and was hybridized topurified ssDNA from the same pool as described. Hybridized moleculeswere then captured by either streptavidin beads (TGFβ1) or streptavidingel shifts (VEGF) and were PCR amplified to generate the next TruncationSELEX pool.

Cloning and Sequencing

RNA recovered from the final-round filters was reverse transcribed andPCR amplified as in every round. The PCR products were purified by PAGelectrophoresis and cloned into the SrfI restriction site of pCR-ScriptDirect SK(+) plasmid using the pCR-Script Amp SK(+) cloning kit(STRATAGENE CLONING SYSTEMS, La Jolla, Calif.). Clones were sequencedwith ABI Prism sequencing kit (Applied Biosystems, Perkin-Elmer, FosterCity, Calif.).

TGF∃1 Bioassay

The bioactivity of TGFμ1 nucleic acid ligands was measured with minklung epithelial cells as described in U.S. patent application Ser.Number 09/046,247, filed Mar. 23, 1998. Briefly, proliferation of thesecells is inhibited by TGFβ1. Human TGFβ1 was titrated on the cells and³H-thymidine incorporation was measured. The point at which ³H-thymidineincorporation by the cells was inhibited by 90-100% was determined(typically 1-4 pM). This inhibitory amount of TGFβ1 along with varyingamounts of nucleic acid ligand (typically 0.3 or 1 nM to 1 or 3 μM, in 3fold increments) was used. Cells were plated at 1×10⁵/ml in 96-wellplates in 100 μl MEM, 10 mM HEPES pH 7.5, 0.2% FBS. Following 4 hours ofincubation at 37° C., when cells were well attached to the well surface,TGFβ1 was added at 1-4 pM with or without nucleic acid ligands asfollows: the ligands were diluted across the 96 well plate in 3-folddilution steps and then TGFβ1 was added at 1-4 pM to all wells exceptcontrols. The cells were incubated for 16-18 hours prior to addition of³H-thymidine, and continued incubation for 20 additional hours following³H-thymidine addition at 0.25 μCi per well. After incubation, the cellswere lysed with 1% Triton X-100 and harvested onto GF/B filter plates bythe Packard 96 well plate harvester, and ³H-thymidine incorporation incellular DNA was quantitated by scintillation counting in microscint atthe Packard Top-Count. Data were plotted as % of max ³H-thymidineincorporation vs RNA concentration and were fitted by the softwareKaleidagraph (Synergy Software, Reading, Pa.) to the equationm3*(m0+m1+(m2)−((m0+m1+(m2))*(m0+m1+(m2))-4*(m0)*((m2))){circumflex over( )}0.5)/(2*(m2)); where m0 is the concentration of competitor RNA; m1is the IC50, m2 is the concentration of TGF∃1, and m3 is the plateauvalue of the fraction of max ³H-thymidine incorporation. Ki values weredetermined from IC₅₀ values according to the equationK_(i)=IC₅₀/(1+([T]/K_(dT)), where [T] is the molar concentration ofTGF∃1 present in the assay and K_(dT) is the concentration of TGFβ1causing 50% inhibition of MLEC proliferation as determined by TGFβ1titration experiments.

EXAMPLE 4

RNaseH Digestion

For application of RNaseH site specific cleavage of RNA to truncationSELEX where 2′F modified nucleotides are present, it is necessary thatRNaseH is able to digest 2′F-deoxyribopyrimidine substituted RNA. Inaddition, in order to eliminate the bulk of the 3′ primer binding site,it would be desirable for the RNaseH to accept targetingoligonucleotides that include the 2′deoxyribonucleotide gap at its3′end.

RNaseH is an enzyme that recognizes RNA-DNA hybrids and digests the RNAstrand through an endonucleolytic mechanism (Hostomsky et al., ColdSpring Harbor Laboratory Press, New York, pp. viii, 499, 1993). RNaseHis a ubiquitous enzyme found in procaryotes and eukaryotes mechanism(Hostomsky et al., Cold Spring Harbor Laboratory Press, New York, pp.viii, 499, 1993). E. coli encodes at least two RNaseH isotypes onseparate genes (Itaya (1990) Proc. Natl. Acad. Sci. U.S.A. 87:8587-91).The E. coli RNaseH1 is most extensively studied. The digestive mechanismof E. coli RNaseH1 is similar to DNases where the 2′OH group does notparticipate in the nucleophilic attack of the phosphodiester bond andthe product ends are 3′OH and 5′P (Crooke et al. (1995) J. Biochemistry312:599-608). It has been shown that E. coli RNaseH 1 could be used forsite directed digestion of RNA using DNA molecules complementary to thesite of digestion (Inoue et al. (1987) FEBS Letters 215:327-330). Thedigestion site could be targeted to a single position using chimerictargeting oligonucleotides containing regions of 2′deoxyribonucleotidesflanked by regions of 2′OMe nucleotides (Inoue et al. (1987) FEBSLetters 215:327-330) taking advantage of the inability of the enzyme todigest RNA-2′OMeRNA hybrids (Inoue et al. (1987) FEBS Letters215:327-330: Lima and Crooke (1997) Biochemistry 36:390-398). The lengthof the 2′deoxyribonucleotide region influences the function of the E.coli RNaseH1 where the cleavage rate is decreased with diminishingnumber of contiguous 2′deoxyribonucleotides, where no cleavage occurringwith less than four contiguous 2′deoxyribonucleotides (Monia et al.(1993) J. Biol. Chem. 268:14514-22; Crooke et al. (1995) J. Biochemistry312:599-608). The site of cleavage by E. coli RNaseH1 of RNA bound tochimeric oligonucleotide containing a 2′deoxyribonucleotide regionflanked by 2′OMe-ribonucleotide regions, has been demonstrated to occurat the 3′site of the RNA base found opposite to the most 5′ nucleotideof the 2′deoxyribonucleotide region (Inoue et al. (1987) FEBS Letters215:327-330; Crooke et al. (1995) J. Biochemistry 312:599-608; Laphamand Crothers (1996) RNA 2:289-296). Cleavage by E. coli RNaseH1 canoccur when the targeting chimeric oligonucleotide contains at least 4contiguous 2′deoxyribonucleotides in the middle or at the 5′end (Inoueet al. (1987) FEBS Letters 215:327-330; Lapham and Crothers (1996) RNA2:289-296), but not at the 3′ end (Inoue et al. (1990) Proc. Natl. Acad.Sci. U.S.A. 87:8587-91) of the targeting chimeric oligonucleotide.Recently, different digestion specificities were found depending on thecommercial source of RNaseH used, where RNaseH from Pharmacia(cat.#27-0894). Sigma (cat. R-6501) or Takarashuzo digest like the E.coli RNaseH1, while RNaseH from Roche Molecular Biochemicals(cat.#786-349) cleaves at one base upstream of the expected site (Laphamet al. (1997) RNA 3:950-951). The effect of 2′F modifications wasdetermined with E. coli RNaseH1 in examples where the modified baseswere at the targeting oligonucleotide. Full 2′F modified targetingoligonucleotides reduced the overall affinity of RNaseH1 for the hybridcomplex with RNA (Crooke et al. (1995) J. Biochemistry 312:599-608).Replacing 2′OMe-deoxyribonucleotide positions of the chimeric targetingoligonucleotide (which include a gap of 2′deoxyribonucleotides in themiddle) with 2′F-2′deoxy-nucleotides reduced the initial cleavage rateby about 5 fold. The effect of 2′F modification of the RNA substrate,instead of the targeting oligonucleotide, was determined only at asingle site, namely at the site of cleavage and found to reduce thereaction rate by 1000-fold (Uchiyama et al. (1994) J. Mol. Biol.243:782-791).

The ability of E. coli RNaseH obtained from Pharmacia and RocheMolecular Biochemicals to cleave 2′F-deoxyribopyrimidine substituted RNAwith chimeric oligonucleotides containing 11 or 122′OMe-deoxyribonucleotides and a gap of 4 contiguous2′deoxyribonucleotides at either their 5′ or 3′ end (FIG. 16) wasdetermined. The data show that only the enzyme from Roche MolecularBiochemicals had significant activity against the tested substrate. Thisenzyme can cleave with either the 5′, the 3′, or both targetingoligonucleotides and it requires the presence of the targetingoligonucleotide for activity. The enzyme from Pharmacia is largelyinactive against the 2′F-deoxyribopyrimidine substituted RNA showingonly slight activity with only the 5′ primer consistent with RNaseH1type activity. We determined the digestion specificity of the enzymefrom Roche Molecular Biochemicals. We used two defined templates(VT30-07, and VT30-44) to generate in vitro 2′F-deoxyribopyrimidinesubstituted RNA transcripts. Such transcripts were end labeled at eitherthe 5′ or the 3′ end, and then treated with RNaseH (from BoheringerMannhein) and the appropriate targeting oligonucleotide so that the 5′and 3′ end labeled RNA was cleaved at its 3′ primer, or 5′ primerbinding site, respectively. Digested RNA was then gel purified andanalyzed on a sequencing gel along with full length RNA and size markersgenerated by alkaline partial hydrolysis of the corresponding endlabeled RNA. Alkaline hydrolysis attacks only the unmodified positionsin the RNA transcripts (2′OH), thus these markers show the positions ofpurines within the transcript. The results are summarized in FIG. 17.The data show that digestion at the 3′ position occurs at a singlephosphodiester bond namely at the 5′ side of the base across the secondbase (from the 5′) of the 2′dexyribonucleotide gap in the 3′ targetingoligonucleotide. Digestion at the 5′ occurs at two positions, namely atthe 5′ sides of the bases across the first and second base (from the 5′)of the 2′dexyribonucleotide gap in the 5′ targeting oligonucleotide. Thedigestion specificity at the 5′ end was confirmed by primer extensionresults (FIG. 18). Full length, and RNaseH digested RNA transcripts, asabove, were used as extension templates of 5′end labeled primerscomplementary to the 3′primer binding site by AMV RT. All extensionreactions were done in the presence of all four2′-deoxyribonucleotide-triphosphates. To facilitate mapping of the 3′endto the template used, additional four extension reactions were done,each containing one of the four chain terminating2′dideoxyribonucleotide-triphosphates, ddATP, ddCTP, ddGTP, and ddTTP.Extended products were then analyzed on sequencing gels as shown in FIG.18. Extension products with all four 2′deoxyribonucleotide-triphosphatesshow not only bands corresponding to the 5′end of the template, but alsobands corresponding to pause sites of RT. The pattern of pause sites isidentical for the corresponding regions of the full length and truncatedtemplates. In agreement with the previous experiment (FIG. 17), thereare two bands corresponding to the 5′ end of the digested RNA, and theshorter bands it does not appear to be a pausing site since there is nocorresponding band in the full length template. As expected, this second5′ end is also present in all four chain termination reactions. Usingthe banding pattern of the chain termination reactions, the 5′ ends ofthe RNaseH digested RNAs could be mapped to the same positions mappedwith the previous experiment. The results from the mapping experimentsat the 5′ digestion site show that the E. coli RNase H from BoheringerMannhein in able to digest at the 3′ phosphodiester bond of a2′F-modified pyrimidine, consistent with the DNase type of reactionmechanism reported for E. coli RNase H1 (Crooke et al. (1995) JBiochemistry 312:599-608). Since the commercial source of RNaseH wasshown to be important in cleavage specificity (Lapham et al. (1997) RNA3:950-951), a panel of 4 RNaseH preparations were tested from differentcommercial suppliers (Epicenter cat#061 00; Stratagene cat#600215; RocheMolecular Biochemicals cat#786357; and MBI Fermentas cat#EN0201) fortheir ability to digest 2′F-deoxyribopyrimidine substituted RNA in thepresence of either the 5′, 3′, or both targeting oligonucleotidesdescribed above. The results in FIG. 19 show that only the enzyme fromRoche Molecular Biochemicals was able to digest efficiently at bothpositions while the rest could digest efficiently only at the 5′position, consistent with RNasH1 type of specificity. All enzymes showedsome trace activity at the 3′ position with the MBI Fermentas lot beingslightly more active.

The data presented here clearly show for the first time, that RNase Hcan digest 2′F-deoxyribopyrimidine substituted RNA at a specific site,directed by an appropriate targeting oligonucleotide. They also show,for the first time, that the Roche Molecular Biochemicals enzyme can usetargeting oligonucleotides containing a gap of four contiguous2′deoxyribonucleotide bases at its 3′ end.

EXAMPLE 5

Biotinylation of RNA

The truncation-SELEX-by-hybridization protocol requires the ability tobiotinylate RNA following RNaseH digestion and affinity selection. Thereare several procedures that allow such RNA modification of which twowere tested with RNaseH digested RNA, namely photo biotinylation andtailing by Terminal Deoxynucleotidyl Transferase (TdT). In addition,biotinylation scheme was developed using RNA ligase. Extent ofbiotinylation was determined by measuring streptavidin complexing ofbody labeled RNA by either gel shift, or capture to streptavidinloaded-beads (Pierce) or -membranes (Promega).

For photobiotinylation and tailing reactions, commercially availablekits (Clontech) were used per manufacturer's instructions. From thesetwo methods only the tailing reaction was successful. By using theenzyme and reaction buffer from Roche Molecular Biochemicals(Indianapolis, Ind.) it was observed that biotinylation efficienciescould be improved. FIG. 20 shows typical biotinylation efficiencies bythe tailing reaction, suggesting that under these experimentalconditions the enzyme is saturated and biotinylates about 30% of theavailable truncated RNA at concentrations as low as 50 nM (0.5 pmoles in10 μl binding reaction). A little higher efficiencies can be obtained(up to ˜45%) by using Biotin-N⁶-ddATP (cat#NEL508, NEN, Boston, Mass.).

As stated above, we developed a new RNA biotinylation method based on T4RNA ligase. In analogy to the RNA labeling reaction utilizing ³²PCP(Cytidine 3′,5′-bis(phosphate), [5′-³²P]), two biotinylation substrateswere used as shown in FIG. 15 obtained by phosphoramidite chemistry(Operon Technologies, Alameda, Calif.). Biotinylation results with twodifferent transcripts (40N7 and TGF∃1 ligand 4003) at two temperatures(4° C. and 0° C.) are shown in FIG. 33. These results show clearbiotinylation reactions only with the longer biotinylation substrateused. To follow the reaction, the biotinylated short oligonucleotideswere kinased using T4 polynucleotide kinase and γ³²P-ATP as describedabove. The presence of biotin was confirmed using streptavidin inducedelectroporetic mobility shifts of gel purified ligation products asdescribed before. Estimated yields of this biotinylation reaction wereabout 80-90%.

EXAMPLE 6

Specificity of Hybridization

The success of the truncation SELEX by hybridization depends on theability of the selected biotinylated truncated RNA to specificallyhybridized to its complementary sequence. The hybridization specificityof an evolved pool (VEGF Rd2), (Ruckman et al. (1 998) J. Biol. Chem.273:20556-67) in the presence of different amounts of excess salmonsperm DNA (FIG. 21) was determined. Briefly, VEGF VT30 Rd 12 RNA wasRNaseH digested to remove 5′ and 3′ fixed regions and biotinylated usingterminal transferase and biotin-dUTP. Biotinylated truncate RNA (0.3pmoles) was mixed with 10 fold excess, 5′-end-labelled ssDNAcomplementary strands and was hybridized overnight at 72° C. in thepresence of 0.1 mM C-TAB in 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA.Reactions with competitor salmon sperm DNA at 1-, 10-, 100-, and 1000-xover biotinylated RNA were also included. The RNA, ssDNA, and salmonsperm DNA were denatured at 95° C. and fast cooled prior to addition tohybridization reaction and incubation at 72° C. Following hybridization,the nucleic acid was recovered by ethanol precipitation, mixed withstreptavidin and analyzed on a native 1×TBE polyacrylamide gel. Hybridformation is apparent in this gel system by a slower electrophoreticmobility of the labeled ssDNA due to complexing streptavidin through thehybridized biotinylated input RNA. The results clearly show thatbiotinylated RNA was able to hybridize to its complement even thepresence of 1000 fold excess nonlabeled competitor DNA.

EXAMPLE 7

Truncation-SELEX-by-Hybrid-Selection

The truncation SELEX by hybrid selection protocol was applied to twopools. The first pool, Round 12 VT30, was derived from the 30N7 randomlibrary, and was selected for binding to VEGF for 12 rounds (Ruckman etal. (1998) J. Biol. Chem. 273:20556-67). The second pool, Round 1240N-TGF∃1, was derived from the 40N7 random library, and was selected tobind TGF∃1 for 12 rounds as described in U.S. patent application Ser.No. 09/046,247, filed Mar. 23, 1998. These pools were selected becauseremoval of fixed regions did not completely eliminate binding to theirrespective targets. FIGS. 22 and 23. Determining the ability of thestarting pools to retain binding after removal of the primer bindingsites might be important because during the SELEX rounds truncatableligands might be eliminated from the population by nontruncatableligands that have better affinities for the target. This was found to betrue with the TGF∃1 pool (FIG. 22) where more advanced pools retainedalmost no binding following removal of their primer binding sites. Asshown in FIG. 22 there is little difference in the binding of the fulllength and truncated Rd12 pool. There is >1000 fold better binding ofthe full length Rd16 pool compared to truncated Rd16 pool, while theRd13 pool showed an intermediate binding.

Affinity selections were done as described in Example 3. The conditionsof affinity selections are summarized in Tables 6A and 6B. The RNA usedat each round was generated by RNaseH digestion as described in Example3. We used two different methods to partition the hybrids followinghybridization. For the VEGF pool, a gel shift method was used on nativegels while for the TGF∃1 pool streptavidin-agarose beads were used asdescribed in Example 3.

FIG. 24 shows typical results with the native gel shift partitionmethod. As seen in FIG. 24A there is a clear streptavidin induced shiftof labeled complementary ssDNA only when streptavidin is used. Weexcised the shifted material from +SA lane, (designated Rd1) and thecorresponding region from the lane where streptavidin was omitted (from−SA lane, designated Rd1-ct). Both gel slices were crushed to recoverany nucleic acid present and eluted material was subject to PCRamplification using the 5P7 and 3P7 primers as described in Example 3.Amplification results, at various numbers of cycles, as shown in FIG.24B indicate that as expected the +SA lane contained more amplifiablenucleic acid since there is a significant amount of product generatedafter 9 PCR cycles. PCR results also show that the native gel partitionmethod has some background since we were able to amplify gel shiftedmaterial from the −SA lane. Both of these amplified pools (Rd1 andRd1-ct) were transcribed and generated RNA was RNaseH digested and usedfor a second round of affinity selection and gel shift partition ofhybridized molecules. As seen in FIG. 24C the +SA lane of the Rd1 poolcontained streptavidin shifted material as expected. However, the −SAlane also contained shifted material suggesting that within one roundmolecules with altered mobility on native gels were selected. Wedesignated such molecules as background. The evolution of “background”was more severe in the Rd1-ct pool presumably due to the sole presenceof such molecules in the −SA lane from the first round of thistruncation SELEX experiment.

FIG. 25 shows typical PCR amplification profiles observed with thestreptavidin-agarose beads partition method. These results suggest goodsignal to noise ratios and during truncation SELEX rounds nonspecificbinding of labeled ssDNA to the streptavidin beads were not observed.

As seen in Tables 6A and 6B improving signal to noise ratios and anincrease of the amount of RNA binding to the target were observedsuggesting the selection process was successful. Pools Rd1-VEGF,Rd1-ct-VEGF and Rd2-TGF∃1 truncation by a hybridization SELEX experimentwere cloned and sequenced.

RNA Sequences from the Truncation SELEX by Hybrid Selection

Twenty four clones from the Rd1-VEGF and Rd1-ct-VEGF were sequenced, and70 clones from the Rd2-TGF∃1 truncation by hybridization SELEX poolswere sequenced. The sequences obtained, and their alignment, suggestedfamily classification, and binding properties are summarized in Tables9-11.

Greater than half of the sequences from the two VEGF experiments couldbe classified into Family 1 and Family 3 identified in the previousSELEX experiment with VEGF (Ruckman et al. (1998) J. Biol. Chem.273:20556-67). The remaining ligands represent orphan sequences althoughsome have some limited homology to ligands from Family 2 of the originalSELEX experiment. Family 2 from the Rd1-VEGF pool contained isolates offrequent ligands from the original SELEX experiment, namely VT30.3 andVT30.1. Interestingly their relative frequencies is opposite from therelative frequency seen in the original SELEX experiment but consistentin their binding phenotype in the presence or absence of their fixedsequences.

Sequences from the Rd2-TGF∃1 truncation by hybridization SELEX pool canbe assigned into ten groups, namely 9 families and a group of orphans.The largest family contains ligands that have been identified asnitrocellulose binding molecules in previous experiments (TGF∃1 patent).The remaining families are rather small and they could be clonalderivatives of unique sequences by PCR mutations. Families 1 and 5resemble families 3 and 1, respectively, from the TGF∃1 SELEX describedin U.S. patent application Ser. No. 09/046,247, filed Mar. 23, 1998.

Binding Properties of Ligands from the Truncation SELEX by HybridSelection

The binding activity of several ligands was determined by nitrocellulosefilter binding, and data are summarized in Tables 9-11.

Binding activities of ligands from the VEGF experiments were compared inthe presence or absence of fixed sequences. Several ligands were alsoanalyzed in the same way from the starting pool (Rd12 VT30) as shown inTable 12. Ligands from all pools included example of both monophasic andbiphasic binding. Each ligand was then scored and classified into fivegroups as follows: (1) ligands that lose significant affinity for VEGFupon removal of their fixed sequences; (2) molecules with affinities forVEGF somewhat affected (positively or negatively) upon removal of theirfixed sequences; (3) molecules that gain significant affinity for VEGFupon removal of their fixed sequences; (4) molecules with affinities forVEGF not affected upon removal of their fixed sequences; and (5)non-binding ligands either with or without fixed sequences. Forquantitative comparison, the data were analyzed in % plateau vs Kdvalues (FIG. 26). For biphasic curves, values of the major affinitycomponent were used. The data points from the full-length ligands definean area representing the average affinity of the full length population.The graphs are divided into four quadrants (I, II, III, and IV)according to this average affinity where quadrant I represents low Kdsand high plateaus (optimum affinities). The remaining three quadrantsrepresent sub-optimal affinities with quadrant IV being the worst. Ifthe analyzed population includes ligands that lose affinity upon removalof the fixed sequences, then affinity points will migrate suboptimalquadrants if affinity measurements were done in the absence of the fixedsequences. The data (FIG. 26) show that the majority of affinity pointsof the starting population occupy the sub-optimum affinity quadrantswhile the majority of affinity points of the selected population occupythe optimum affinity quadrants. As expected the control population showsa distribution similar to the starting population. Comparison of thefrequencies of ligands in each quadrant is shown in FIG. 27. These dataclearly show that Truncation SELEX by hybrid selections, within oneround, shifted the population frequencies so truncatable ligands becomemore abundant.

Since in the original TGF∃1 SELEX we did not isolate any truncatableligands, finding any truncatable ligand in the truncation SELEX poolswould be an indication of the successful application of the methodsdescribed here. The binding of the ligands from the TGF∃1 truncationSELEX was determined only after RNaseH digestions and the data aresummarized in Table 11. More than half (52%) of ligands tested bound thetarget with high affinity in the absence of fixed sequences. Samplebinding curves are shown in FIG. 28. Truncation SELEX did not onlyidentified truncatable nucleic acid ligands that can bind TGF∃1 but alsonucleic acid ligands that could be TGF∃1 inhibitors. FIG. 31 shows theinhibition of TGF∃1 by ligand 70 from the TGF∃1 Rd2 truncation SELEX byhybridization. This ligand causes full inhibition with an approximate Kiof 74.6 nM while a control sequence show no inhibition with an estimatedKi of worse than 1000 fold compared to ligand 70.

EXAMPLE 8

Ligation of 3′ Primer Binding Sequence

One of the essential steps of the truncation SELEX by ligation processis the introduction of the 3′ fixed sequence at the 3′ end of theselected RNA by ligation. The ability of T4 RNA ligase to catalyze sucha reaction utilizing RNA transcripts ,generated by two differenttemplates (35N-Nae and 35N-Bsm) was determined as shown in FIG. 34.Before transcription, the templates were digested by the correspondingrestriction enzyme (BsmI or NaeI). Transcription was done with α-³²P-ATPand 2′F-pyrimidine-triphosphates to generate body labeled 2′F modifiedtranscripts. In addition, the ability of bridging complementaryoligonucleotides to improve reaction yields was tested. Oligonucleotidesused in this ligation experiment are shown in Table 13. Ligationoligonucleotides were designed to include three biotin residues at their3′ end to allow the measurement of the ligation efficiency by gel shiftand were also synthesized with phosphates at their 5′ end. Bridgingoligonucleotides were designed to be complementary to the 5′ end of theligation oligonucleotide and to include a 6N random region at their 3′ends to allow hybridization at the RNA template thus helping in aligningand holding the two participating templates (the RNA and the 3′ fixedsequence oligonucleotide) in juxtaposition to facilitate their ligation.Oligonucleotides 3G7RC and 3G7R6 were used with the 35N-Nae RNA while3LBsmC and 3LBsm with the 3N-Bsm RNA. Ligation reactions were set asdescribed in the materials and methods and ligation efficiency wasdetermined by quantitation of the residual transcript (TR) and theligated product (LP) as shown in FIG. 34. Control lanes showingstreptavidin induced gel shift also confirmed correct ligation products.In these control lanes, the only way that streptavidin could cause gelretardation of the body labeled transcripts would be by the action of T4RNA ligase causing the covalent attachment of the biotinylatedoligodeoxynucleotide to the labeled RNA. 66% and 55% ligation efficiencywas observed for the 35N Nae RNA in the absence and presence of thebridge oligonucleotide, respectively. Similarly, the ligation efficiencyfor the 35N Bsm RNA was 78% and 41% in the absence and presence of thebridge oligonucleotide, respectively. These results clearly demonstratethat T4 RNA ligase can very efficiently add 3′ fixed sequences to 2′Fmodified RNA in the absence of a bridging oligonucleotide. This methodwas routinely used in the truncation SELEX by ligation scheme.

Ligation of 5′ Primer Binding Sequence

A second essential step of the truncation SELEX by ligation process isthe introduction of the 5′ fixed sequence to the selected RNA. There aretwo ways that this can be accomplished. One way is to attach the 5′fixed sequence to the 5′ end of the selected RNA using T4 DNA ligase anda bridge oligonucleotide. The second way relies on the same type ofreaction where the complement of the 5′ fixed sequence is attached tothe 3′ end of the cDNA copy of the selected RNA. This cDNA copy isgenerated following ligation of the 3′ fixed sequence as described aboveand RT reaction using a primer complementary to the introduced 3′ fixedsequence. Both of these approaches were tested (FIG. 35).

For ligation at the 5′ end of 2′F modified RNA, lightly body labeledtranscript generated as above on the 35N Bsm template was used. Thistranscript was then phosphatased by calf intestinal alkaline phosphatase(Roche Molecular Biochemicals, cat#1097075) as described above to removethe phosphate structures generated by transcription initiation (usuallya tri- or di-phosphate). The phosphatased RNA was then kinased with T4polynucleotide kinase and y³²P-ATP as described above and then used as asubstrate in ligation reactions with T4 DNA ligase from two vendors(Roche Molecular Biochemicals, cat#0481220 and New England Biolabs,cat#202S) containing the oligonucleotide 5G7NOT7 and the bridgeoligonucleotide 5G7RC at 20 and 50 fold excess over the RNA,respectively (sequences of DNA oligonucleotides used are shown in FIG.35. Ligation reactions were set at different concentrations of 2′Fmodified RNA (two of which are shown in Figure 35 and were incubated at4° C. for 16 hours. Following ligation, the reaction products wereanalyzed on denaturing (7 M urea) 10% polyacrylamide gels. The dataclearly show that under these reaction conditions ligation occurs withat least 50% efficiency at RNA concentrations 25-50 nM and both enzymelots were equivalent.

For ligation at the 3′ end of the cDNA, 2′F RNA transcripts from VT30round 12 pool were reverse transcribed using appropriate primer in thepresence of α-³²P-dCTP (NEN, 800 Ci/mmol cat#BLU013A). Labeled cDNA wasligated to preannealed 5G7RC Linker/5′N7 or 5N7 Linker/5N7 bridge asdescribed in Example 3 and ligation products were analyzed on denaturing(7M urea) 10% polyacrylamide gels. The linker oligonucleotides were in2- or 5-fold excess over the cDNA while the bridge oligos were at 3- or7.5 fold excess. The data shown in FIG. 35 show that ligation reactionsoccurred with varied efficiencies. The set that included a fixedsequence bridge had better yields of about 42% and 46% for the 1:2 and1:5 ratio of cDNA to linker oligonucleotide, respectively. The ligationefficiencies with the bridge sequence containing 5 random positions wereabout 15% at the 1:5 ratio of cDNA to linker oligonucleotide. Duringtruncation by ligation SELEX rounds we used the ligation to the cDNAmethod to introduce the 5′ fixed sequence to the truncated RNA since theligation at the 5′ end of the RNA would require additional enzymaticmanipulations at each step, namely phosphatase treatment and kination.

Truncation SELEX by Ligation

We applied the truncation SELEX by ligation protocol to three poolsnamely, the same two pools used for the truncation SELEX by hybridselection protocol and in addition the round 12 30N-TGF∃1pool.

For generating the 3′ fixed sequence we used oligo 3G7RC (Table 13) forthe VEGF pool while 3G7RCD was used for the TGF∃1 pools. The reason forusing 3G7RCD was to avoid repetitions of the sequence 5′CA at the end ofthe library with each SELEX round due to the RNaseH digestion leavingbehind the CA sequence from the fixed sequence. To ensure betterefficiency at each round, the starting library was modified by removinga significant portion of 5′ fixed sequence leaving behind the 5′GGGAGtranscription initiation site as follows. RNA transcripts from each ofthe starting pools were RNaseH digested at their 5′ ends, reversetranscribed using a primer complementary to the 3′ fixed sequence, andresulting cDNAs were ligated to the 5N7-Linker/5N7-Bridge set of oligos(FIG. 35) as described above. This ligation (even though inefficient)generates molecules that have their 5′ fixed sequence replaced with theT7 promoter-5′GGGAG sequence fused to the RNaseH digestion site. Thesenew templates generate RNA containing just 5′GGGAG as fixed sequence.These RNA molecules do not require RNaseH digestion at the 5′ end priorto selection and the 5′ primer binding sequence can be introduced(following selection, 3′ ligation and reverse transcription) using the5G7RC-Linker/5′N7 oligonucleotide set (FIG. 35) which showed betterligation efficiencies compared to the 5N7-Linker/5N7-Bridge set. FIG. 36shows that as expected, PCR products of the 5′ truncated VEGF pools areshorter than the starting round 12 pool. In addition, the 5′ truncatedstarting pool retain some binding to VEGF compared to random RNA (FIG.36).

At each round, affinity selections were done as described in Example 3.The conditions of affinity selections are summarized in Tables 6C and6D. The RNA used at each round was generated by RNaseH digestion at the3′ fixed sequence as described in Example 3.

As seen in Tables 6C and 6D improving signal to noise ratios and anincrease of the amount of RNA binding to the target was observedsuggesting the selection process was successful. The starting 5′truncated starting VEGF pool (designated T1), VEGF round 1 (designatedT2), VEGF round 2 (designated T3), TGF∃1-30N round 2, and TGF∃1-40Nround 2 pools were cloned and sequenced. The pools from these roundswere chosen for cloning and sequencing based on their bindingproperties.

EXAMPLE 9

RNA Sequences from the Truncation SELEX by Ligation

With the Truncation SELEX by Hybridization protocol it was easy todetermine the effect of the selection process on the ligand frequencywith respect to the effect of the fixed sequences on their bindingactivity. This was feasible since the cloned ligands contained bothfixed sequences. With the Truncation SELEX by Ligation protocol as itwas applied here, this was not feasible because the starting pool wastruncated at the 5′ end removing the bulk of the 5′ fixed sequence. Todetermine the effect of the ligation protocol on the phenotype frequencyof selected VEGF ligands, the starting 5′ truncated pool as well as thepools from round 1 and round 2 were cloned and sequenced. 43, 22, and 21ligands were sequenced from the starting 5′truncated, round 1 and round2 VEGF pools, respectively. With the TGF∃1 experiment, the ability toidentity truncatable molecules was of interest and not in the comparisonof phenotype frequencies. Therefore, only the final pools, namely round2 from both the 30N and 40N experiment were sequenced.

The obtained sequences, their alignment, suggested familyclassification, and binding properties are summarized in Tables 14-18.

The starting 5′ truncated VEGF pools contained ligands that could beassigned into the three families identified previously (Ruckman et al.(1998) J. Biol. Chem. 273:20556-67) and into a fourth family and a groupof orphan sequences pyrimidine rich at their 3′ ends. The conservedsequence found at the 5′ end of members of family 4 is identical to thesequence of the 3′ fixed sequence and most likely this family mightrepresent an artifact of the ligation reactions. As with the unmodifiedVT30 pool, members of family 2 are rare with only one example found. Themajority of sequences in this starting pool contain the 3′ fixedsequence 5′CA probably a remnant of the 3′ terminal sequence removedduring the process of construction of this pool. In both of the selectedVEGF pools, the frequency of the first three families is increasedcompared to the starting pool especially for family 2 while thefrequency of the orphan sequences was decreased. The selected pools alsocontained versions of ligand VT30.3 with additional sequences at bothends. Ligand VT30.3 was found in high frequency in the first SELEXexperiment (Ruckman et al. (1998) J. Biol. Chem. 273:20556-67). Themajority of ligands from the round 2 pool contain multiple copies of thesequence 5′CA at their 3′ ends, presumably a remnant of the RNaseHdigested 3′ fixed sequence, which was added with each ligation step andleft behind after each RNaseH digestion.

Sequences from the Rd2-30N TGF∃1 truncation by ligation SELEX pool canbe assigned into three groups, namely 2 families and a group of orphans.The orphan group contains ligands that have been identified asnitrocellulose binding molecules in previous experiments described inU.S. patent application Ser. No. 09/046,247, filed Mar. 23, 1998. Theremaining two families contain several highly homologous members thatcould be clonal derivatives of one or a few unique sequences by PCRmutations. Family 2 contains ligands that were identified at very lowfrequency in the first TGF∃1 SELEX experiment described in U.S. patentapplication Ser. No. 09/046,247, filed Mar. 23, 1998. Namely, there is agroup of sequences (20, 27, 19, 12, 34, and 3) that share a large blockof common sequence in the majority of the molecule differing only by twobases (5′CA) at their 3′ end and 1-8 bases at their 5′ end possibly dueto ligation artifacts. Their common sequence is identical to thesequence of ligand 30-32 isolated in our first SELEX experiment withTGF∃1 described in U.S. patent application Ser. No. 09/046,247, filedMar. 23, 1998. Ligands from family one are highly conserved (and couldbe derivatives of a single sequence found in the starting random RNApool) and were not isolated before. Since we use DNA oligo without theCA sequence at its 5′ end for ligation at the 3′ end o′ TGF∃1 selectedRNA, the isolated ligands from the Rd2-30N TGF∃1 truncation by LigationSELEX pool do not contain the sequence 5′CA at their 3′ ends.

Sequences from the Rd2-40N TGF∃1 truncation by ligation SELEX pool canbe assigned into seven families. These families are rather small andthey could be clonal derivatives of one or a few unique sequences by PCRmutations. The last family contains ligands with sequencescharacteristic of nitrocellulose binders. All the ligands from family 1contain the 3′ fixed sequence of the N7 series at their 5′ end and themajority of them contain at their 3′ ends a large portion of the T7promoter. Thus, these ligands must represent artifacts of the ligationreactions. Family two contains ligands of the same class as ligands fromfamily 2 of the Rd2-30N TGF∃1 truncation by ligation SELEX pool but themajority of them contain the 3′ fixed sequence at their 5′ ends.Therefore they could be contaminants from the 30N pool that weremodified due to undesirable ligation side reactions. Family 2, however,contains three ligands that could be true isolates from the 40N pool.The other families contain ligands that were not identified in U.S.patent application Ser. No. 09/046,247, filed Mar. 23, 1998. Family fourcontains members that are longer than expected by ten bases. As with theligands from the 30N pool, the ligands from the 40N pool do not containthe sequence 5′CA at their 3′ end, but unlike the 30N ligands the 40Nligands have a somewhat heterogeneous 5′ initiator sequence.

EXAMPLE 10

Binding Properties of Ligands from the Truncation SELEX by Ligation

The binding activity of several ligands from the Truncation SELEX byLigation experiments was determined by nitrocellulose filter binding anddata are summarized in Tables 14-18.

Binding activities of ligands from the VEGF experiments were determinedwith or without the 3′ fixed sequence. Ligands from all pools includedexamples of both monophasic and biphasic binding. Example binding curvesare as shown in FIG. 37. Like the truncation SELEX by hybridizationexperiment, each ligand was scored and classified into five groups asfollows: (1) ligands that lose significant affinity for VEGF uponremoval of their 3′ fixed sequences; (2) molecules with affinities forVEGF somewhat affected (positively or negatively) upon removal of their3′ fixed sequences; (3) molecules that gain significant affinity forVEGF upon removal of their 3′ fixed sequences; (4) molecules withaffinities for VEGF not affected upon removal of their 3′ fixedsequences; and (5) non-binding ligands either with or without the 3′fixed sequences. For quantitative comparison, the data were analyzed asabove in % plateau vs Kd values (FIG. 38). For biphasic curves, valuesof the major affinity component were used. Because the members of thestarting pool for this SELEX experiment were truncated at their 5′ ends,the phenotype of ligands as full-length molecules containing both the 5′and 3′ fixed sequences could not be compared. In order to define theaffinity quadrants, the affinity points of full-length ligands from theRd12 VT30 pool were used. Therefore, based on the affinity values of theRd12 VT30 ligands, the graphs are divided into four quadrants (I, II,III, and IV) where quadrant I represents low Kds and high plateaus(optimum affinities). The remaining three quadrants representsub-optimal affinities with quadrant IV being the worst. In all poolsfrom this truncation SELEX by ligation experiment, the ligandscontaining the 3′fixed sequence occupied suboptimal affinity quadrants(FIG. 38). Upon removal of the 3′ fixed sequence, the majority of theaffinity points of the selected pools (but not of the 5′ truncatedstarting pool) occupied the optimum affinity quadrant. Comparison of thefrequencies of ligands in each quadrant is shown in FIG. 39. These dataclearly show that truncation SELEX by ligation, within one round,shifted the population frequencies so truncatable ligands become moreabundant. Of interest is the observation that in the presence of the 3′fixed sequence the majority of ligands tested showed worse affinitiessuggesting that the 3′ fixed sequence probably influences the folding ofthese ligands towards incorrect conformations.

As with the truncation SELEX by hybridization experiment, the binding ofthe ligands from the TGF∃1 truncation by ligation SELEX was determinedonly after RNaseH digestions to remove the 3′ fixed sequence and thedata are summarized in Tables 17 and 18. The majority of selectedligands tested bound the target with high affinity in the absence of the3′ fixed sequence. Sample binding curves are shown in FIG. 40.

EXAMPLE 11

Truncation SELEX with DNA Libraries

The truncation SELEX process described herein is designed for use withRNA (native or modified) libraries. With slight modification, thisapproach can be applied to ssDNA libraries. These modifications could beapplied to templates engineered to include restriction enzymerecognition sites at the junction of the random region with the 5′ and3′ fixed sequences (FIGS. 32 and 41). The ssDNA library lacking the 5′and 3′ fixed sequences can be generated in at least three ways (FIG.32). First, sense strands can be purified using a biotinylated 3′ primerand streptavidin induced gel shift on denaturing gels (FIG. 32, leftbranch) (Pagratis (1996) Nucleic Acids Research 24:3645-6). Purifiedsense strands can then be annealed to single stranded oligoscomplementary to the 5′ and 3′ fixed sequences containing therestriction sites (sequences could be as shown in FIG. 41. Annealedoligonucleotides are then digested with the appropriate restrictionendonuclease and the fragment containing the random region is purifiedby denaturing gel electrophoresis. Second, double stranded PCR templatescan be digested with appropriate restriction endonuclease to remove the5; and 3′ fixed sequences (FIG. 32, middle branch). In this case,restriction sites are chosen to generate a 3′ recessive and a 5′recessive end at the 5′ and 3′ fixed sequences, respectively. Followingdigestion the 3′ recessive end is filled-in with the Klenow fragment ofE. coli DNA polymerase-I and biotin-dUTP. The non-biotinylated sensestrand is then purified by streptavidin induced gel shift on denaturinggels (Pagratis (1996) Nucleic Acids Research 24:3645-6). Third, in avariation of the second method, the double stranded PCR templates can bedigested with appropriate restriction endonuclease to remove first the5′ fixed sequence. In this case restriction sites that generate a 3′recessive end can be used at the 3′ fixed sequence. Following digestionat the 5′ fixed sequence, the products are biotinylated as above withthe Klenow fragment of E. coli DNA polymerase-I and biotin-dUTP.Following biotinylation, the 3′ fixed sequence is digested away usingthe appropriate restriction enzyme and the nonbiotinylated sense strandis purified as above (Pagratis (1996) Nucleic Acids Research 24:3645-6).Following target partition selected ssDNA molecules can be amplified byeither following the hybrid selection approach or the ligation approach.For the hybrid selection approach, a portion of the selected pool fromthe previous round is amplified using a biotinylated 5′ primer and thenonbiotinylated antisense strands are purified as above (Pagratis (1996)Nucleic Acids Research 24:3645-6). The selected RNA is then biotinylatedas in the case of RNA (for example with terminal transferase andBiotin-dUTP) and hybridized in excess of purified full length antisensestrand in the presence of nonspecific competitor (as described abovewith the RNA truncation SELEX by hybridization method). Hybridizedmolecules are then captured as before (for example streptavidin beads)and PCR amplified to generate the pools for the next round. For theligation approach, the selected ssDNA is ligated to primers in thepresence of appropriate bridging oligonucleotides or alternative usingappropriately designed stem-loop structures (FIG. 41).

The data presented in Examples 3-11 clearly demonstrate the feasibilityof isolating nucleic acid ligands that utilize just the random regionfor high affinity binding to their target. To isolate such nucleic acidligands it is not necessary to start with unselected pools. Whenstarting with evolved pools, selection could happen relatively fastwithin 1-2 rounds. Two different approaches were described, one based onhybrid selection of complementary full-length molecules and the otherbased on enzymatic generation of primer binding sites to allow PCRamplification. Both methods gave similar results. FIG. 42 shows theprogress of both selection processes showing almost superimposibleresults. FIG. 43 compares the binding phenotype of individual ligandswith or without their fixed sequences. Within one round of selection thetwo processes described here dramatically increase the frequency ofligands that bind the same or better to the target if their fixedsequences are removed. The shift in frequencies with the ligation methodis more pronounced suggesting a more efficient selection. Although theligation method gave more striking results, it was found that thismethod yielded artifact ligands that resulted from the shuffling offixed sequences by the enzymatic steps used. Such sequencerearrangements were not found with the hybrid selection method.

EXAMPLE 12

Screening for Ligands of a Certain Size

The experimental demonstration of the method shown in FIG. 5 isdescribed in this example. Body labeled KGF ligands 56F, 53F, 38F, 26F,15F and 14F were cleaved by alkaline hydrolysis, bound to KGF and thebound fragments were partitioned by nitrocellulose filtration andanalyzed on a sequencing gel (FIG. 12). The potential minimal fragmentsare marked on the gel suggesting that ligands 53F, 38F, 26, 15F, and 14Fshow minimal possible sizes in the 45 base range.

Minimum size fragments can also be identified in pools of moleculescontaining truncatable ligands to reasonable frequencies. As shown thereconstruction experiment in FIG. 13, alkaline hydrolysis followed byaffinity selection, allows the identification of the minimum fragment aspredicted by conventional truncation experiments with approximate sizeof 59 bases. Mixing of K14 ligand with random RNA at rations of p to1:100, random fragmentation and affinity selection allows theidentification of the expected K14 minimum fragment.

Alkaline hydrolysis is routinely used to create the random cleavageladders, but it creates homogeneous ladders only with RNA. Modified RNAat the 2′ position of pyrimidines (lives sequence specific incompleteladders while DNA is not susceptible to alkaline hydrolysis. We tested agroup of nucleases for their ability to generate random complete ladderswith 2′F-pyrimidine modified RNA. Enzymes tested were mung beannuclease, nuclease S1, snake venom phosphodiesterase, nuclease S7, andnuclease P1. Only snake venom phosphodiesterase, nuclease S7, andnuclease P1 resulted in homogeneous laddering of the oligonucleotideused (FIG. 14). Since snake venom phosphodiesterase is an active 3′ to5′ exonuclease with some endonuclease activity only the nuclease P1 andS7 can be used in minimum fragment determination experiments. TABLE 1(SEQ ID NO:1) 5′-gggaggacgaugcgg-GENOMIC INSERT-cagacgacgagcggg a-3′   *******************         ******************* 3′-ccctcctgctacgcc-5′(SEQ ID NO:2) 3′-gtctgctgctcgccct-5′ (SEQ ID NO:3)

TABLE 2 Isolates with the consensus binding site from SELEXes withchanging fixed sequences. SEQ # Iso- ID Sequence of the isolate's insertCopies late NO: Location in E.coli genome 3+4 5 1 4 GGCACGCGUAUUCA CUG

GC AUCA GC CAG 58 4 ACUGUGU 2 sense of rffG (dTDP-D-glucose 4,6dehydratase; participates in forma- tion of lipopolysaccharide in thebacterial outer membrane) 210 nt from start codon 2 5CUUCGCUUCAGUACCGUCG AGC

GC ACCA GC 5 — GUU UCGCC antisense of phe T (phenylalanyl- tRNAsynthetase), 250 nt from start codon 3 6 AAUGU UCC

CC AGCA GG GGA 7 7 AAUGAUGUUGUUCUGGCU antisense of o356 (similarity tolpfD from S. typhimurium fimbrial operon), 140 nt from stop codon;antisense upstream of o180 (similarity to fiml from S. 4 7UAGGAGAGCGUUAAC AAC

GC AUCA GC GUU 1 — GAAGUGACGGAGGU antisense of ygil (hypotheticaltransmembrane protein), 470 nt from start codon 5 8 UGAUU UGC

GC AACA GC GCA 1 1 AUGAGGAAAGAGAGCCAGAUUACCC antisense of mreD(responsible for formation of the rod shape of E. coli cells, integralmembrane protein, 50 nt from start codon 6 9 UGACUG GAC

UC AUCA GA GUU UGCACUU 1 — GAG antisense of secY (membrane proteinessential for protein export), 60 nt from stop codon 7 10 AGUAAUUAC GCC

UG AACA CA GGC 1 — AUAAAGAAGUACAUAUGGU not in GenBank 8 11UAUUCUGGCUAUCUACA GCU

CC AGCA GG 1 — AGC UGGAGAUCAACGAUCCU sense of ebgR (ebg (beta-galactosidase) repressor), 210 nt from start codon 9 12UGAGUGCCGAAGAUCGUGAG CAG

GG AACA — 1 CC CUG AUUAUCC not in GenBankThe consensus binding site elements (FIG. 1A) are separate by spaces.The RNYA loop is in boldface, the bulged

is

, the 2 nucleotide stem is doubled-underlined, and the3 nucleotide stem is underlined. Isolate no. 1 is shown folded in FIG.1d. “# copies” indicates in how many copies a particular isolate wasfound, out of the total 101 isolates sequenced in SELEX experiments 3and 4, and out of 72 isolates# in SELEX experiment 5 (FIG. 3).

TABLE 3 Matches found in E. coli genome to the structure of SELEXconsensus MS2 CP binding site, using RNAMOT program. SEQ ID Binding SiteNo. NO: Location in E. coli genome  1 13 CCG

CG ACCA CG CGG antisense of ybdH (some similarity to glyceroldehydrogenates), 20 nt from start codon; sense upstream of o386(similarity to YJG0_YEAST, which is a hypothetical amino- trasferase),130 nt from start codon  2 14 ACG

CC AUCA GG CGU antisense of jltJ (glutamate/aspartame transport system(membrane-bound) permeate protein GltJ), 90 nt from start codon;antisense downstream of ybeJ 60 nt from stop codon  3 15 CUG

CG AGCA CG CAG antisense of ORF F486 (similarity to YC39_CYAPA), 400 ntfrom start codon  4 16 ACCAGCACCAGCGGU antisense of bglX (periplasmicbeta- glucosidase precursor), 660 nt from stop codon  5 17 GCC

GC AACA GC GGC antisense of nuoN (putative membrane protein, chain N ofNADH dehydrogenate I (multisubunit membrane protein)), 740 nt from stopcodon  6 18 UGC

GC AGCA GC GCA antisense of nuoK (putative membrane protein, chain K ofNADH dehydrogenate I (multisubunit membrane protein)), 60 nt from stopcodon, 230 nt from start codon  7 19 CCC

GC AACA GC GGG antisense of yfcA (potential integral membrane protein),30 nt from start codon  8 20 CAC

GC AUCA GC GUG antisense of ORF f848 (similarity to PBPA_HAEIN, which ispenicillin-binding protein 1A, potential inner membrane protein), 220 ntfrom stop codon  9 21 UGC

GC AACA GC GCA antisense of mreD (responsible for formation of the rodshape of E. coli cells, integral membrane protein), 50 nt from startcodon 10 22 CUG

GC AUCA GC CAG sense of rffG ((dTDP-D-glucose 4,6 dehydratase;participates in formation of lipopolysaccharide in the bacterial outermembrane), 210 nt from start codon 11 23 GCC

GC AACA GC GGC antisense of yigR, 350 nt from start codon 12 24 GUG

CC AACA GG CAC antisense of yhiN, 250 nt from stop codon 13 25 GCC

GC AGCA GC GGC sense of fic (involved in cell division; filamentationand induction of a membrane protein in presence of cyclic AMP inmutant), 80 nt from start codon; sense downstream of yhfG, 70 nt fromstop codon 14 26 UGC

CC AUCA GG GCA antisense of dacB (penicillin-binding protein 4,DD-carboxypepidase 1B, cyto- plasmic membrane protein), 110 nt from stopcodon 15 27 UCC

GC ACCA GC GGA antisense of agaZ (putative tagatose 6- phosphatekinase), 80 nt from stop codon; sense upstream of agaR (putativetranscrip- tional repressor of aga operon for N-acetyl- galactosaminetransport and metabolism), 330 nt from start codon 16 28 ACC

GC AUCA GC GGU antisense of ygiT (probable integral membrane protein),340 nt from stop codon 17 29 GGC

GG AACA CC GCC antisense of o1025 (similarity to acrF, which is anintegral membrane protein involved in cell division (cell envelopeformation and multidrug resistance), 1330 nt from strat codon 18 30 UCG

CC ACCA GG CGA antisense of topA (alternate name supX, DNA topoisomeraseI, omega protein I), 600 nt from stop codon 19 31 UCC

CC AGCA GG GGA antisense of o356 (similarity to lpfD from the S.typhimurium fimbrial operon), 140 nt from stop codon; antisense upstreamof o180 (similarity to fimI from S. typhimurium, which is afimbriate-related fibrin-like protein), 150 nt from start codon 20 32GCC

GC AUCA CC CCC sense of tesA (alternate gene name apeA, acyl-CoAthioesterase I, periplasmic enzyme, involved in membrane lipidbiosynthesis), 200 nt from start codon, 420 nt from stop codon;antisense upstream of ybbA (hypothetical ABC transporter), 160 nt fromstart codon 21 33 GGC

GC AUCA GC GCC sense of yagX (similarity to cfaC, which is colonizationfactor antigen 1 fimbrial subunit C precursor, may serve as anchor forthe fimbriate in the outer membrane). 1170 nt from stop codonThe sites shown double-underlined and in boldface (9, 10 and 19)correspond to SELEX isolates 5, 1 and 3 in TABLE 1. The consensusbinding site elements (FIG. 1E) are separated by spaces. The ANCA is inboldface, the bulged

is

, the 2 nucleotide stem^(double-underlined, and the 3 nucleotide stem is underlined.)

The consensus binding site elements (FIG. 1E) are separated by spaces.The ANCA is in boldface, the bulged A is shadowed the 2 nucleotide stemis double-underlined, and the 3 nucleotide stem is underlined. TABLE 4SEQ ID NO: Set One 3′-ApTpTpApTpGpCpTpGpApGpTpGpApTpApTp 5G7 RC Linker34  ||||||||||||||||| 5′-TpApApTpApCpGpApCpTpCpApCpTpApTpApGpGpGpA 5′N735 Set Two 3′-ApTpTpApTpGpCpTpGpApGpTpGpApTpApTpCpCpCpTpCp 5N7 Linker 36   | | | | | | | | | | | | | | | | | | | | | |5′-TpApApTpApCpGpApCpTpCpApCpTpApTpApGpGpGpApGpNpNpNpNpN-3′ 5N7 Bridge37

Bridge/Linker sequences used to ligate at the 3′end of truncated cDNA.Set two was used to constract the starting library only while set oneTABLE 5 SEQ ID NO: Starting DNA templates: 40N 7:5′GGGAGGACGATGCGG[-40N-]CAGACGACTCGCCCGA 3′ 38 30N7:5′GGGAGGACGATGCGG[-30N-]CAGACGACTCGCCCGA 3′ 39 SELEX PCR Primers: 5G7:5′TAATACGACTCACTATAGGGAGGACGATGCGG 3′ 40 3G7: 5′TCGGGCGAGTCGTCTG 3′ 41

TABLE 6A VEGF Truncation-by-hybridization SELEX SELEX-Rd [P]¹, nM [R]²,nM % B³ S/N⁴ PF⁵ PB⁶ Bf.Wsh⁷ U.Wsh⁸ Used⁹ Rd1 0.200 1.00 64.0 85.0 + −20 ml — comb. 0.040 0.20 22.5 25.0 + − 20 ml — 0.008 0.04 5.0 5.50 + −20 ml — Rd2 0.040 0.20 77.0 37.50 + − 20 ml — comb. 0.008 0.04 75.018.00 + − 20 ml —¹Protein concentration in nanomolar²RNA concentration in nanomolar³Bound RNA expressed as % of max⁴Signal to noise⁵Use of nitrocellulose prefiltered RNA⁶Use of preblocked nitrocellulose with BSA⁷Volume in ml of buffer wash⁸Volume in ml of 0.5M urea wash (— indicates no urea wash)⁹Pool carried forward to the next round (comb. means that the pools werecombined before taken to the next round)

TABLE 6B TGF∃1 Truncation-by-hybridization SELEX U. SELEX-Rd [P^(]1), nM[R]², nM % B³ S/N⁴ PF⁵ PB⁶ Bf. Wsh⁷ Wsh⁸ Used⁹ 40N-Rd1 1.0 5.0 1.918.70 + +  5 ml 10 ml comb. 0.3 1.5 1.5 9.00 + +  5 ml 10 ml 40N-Rd2 1.05.0 4.8 14.73 + + 10 ml  5 ml comb. 0.3 1.5 4.4 34.75 + + 10 ml  5 ml40N-Rd3 0.3 1.5 4.0 8.00 + + 10 ml  5 ml 40N-Rd4 0.3 1.5 3.0 1.50 + + 10ml  5 ml stopped 0.1 0.5 2.0 3.30 + + 10 ml  5 ml¹Protein concentration in nanomolar²RNA concentration in nanomolar³Bound RNA expressed as % of max⁴Signal to noise⁵Use of nitrocellulose prefiltered RNA⁶Use of preblocked nitrocellulose with BSA⁷Volume in ml of buffer wash⁸Volume in ml of 0.5 M urea wash⁹Pool carried forward to the next round (comb. means that the pools werecombined before taken to the next round)

TABLE 6C VEGF Truncation-by-ligation SELEX SELEX-Rd [P]¹, nM [R]², nM %B³ S/N⁴ PF⁵ PB⁶ Bf.Wsh⁷ U.Wsh⁸ Used⁹ Rd1 0.2000 1.000 37.0 32 − +  8 ml— yes 0.0400 0.200 18.5 26 − +  8 ml — yes 0.0080 0.040 4.0 80 − +  8 ml— no Rd2 0.2000 1.000 108.5 166 − + 25 ml — no 0.0400 0.200 65.5 98 − +25 ml — yes 0.0080 0.040 15.5 ND¹⁰ − + 25 ml — no Rd3 0.2000 1.000 103.5192 − + 25 ml — — 0.0400 0.200 92.0 204 − + 25 ml — — 0.0080 0.040 21.051 − + 25 ml — — 0.0016 0.008 6.0 10 − + 25 ml — —¹Protein concentration in nanomolar²RNA concentration in nanomolar³Bound RNA expressed as % of max⁴Signal to noise⁵Use of nitrocellulose prefiltered RNA⁶Use of preblocked nitrocellulose with BSA⁷Volume in ml of buffer wash⁸Volume in ml of 0.5M urea wash⁹Pool carried forward to the next round (comb. means that the pools werecombined before taken to the next round)¹⁰Not determined

TABLE 6D TGF∃1 Truncation-by-ligation SELEX U. SELEX-Rd [P]¹, nM [R]²,nM % B³ S/N⁴ PF⁵ PB⁶ Bf. Wsh⁷ Wsh⁸ Used⁹ 40N-Rd1 1.000 5.000 3.75 2.1 −10 ml  10 ml — yes 0.330 1.650 2.30 1.2 − 10 ml  10 ml — no 0.110 0.5500.30 <1.0 − 10 ml  10 ml — no 40N-Rd2 1.000 5.000 10.40 3.3 + 2 ml 10 ml— yes 0.330 1.650 4.20 1.4 + 2 ml 10 ml — no 0.110 0.550 0.25 1.0 + 2 ml10 ml — no 40N-Rd3 0.330 1.650 4.50 2.2 + 2 ml 10 ml — yes 0.110 0.5501.80 1.4 + 2 ml 10 ml — no 0.033 0.165 0.30 1.1 + 2 ml 10 ml — no40N-Rd4 0.330 1.000 11.20 29.1 + 2 ml 10 ml — 0.110 0.330 5.50 13.5 + 2ml 10 ml — 0.033 0.100 1.80 5.5 + 2 ml 10 ml — 30N-Rd1 1.000 5.000 2.006.8 − 10 ml  10 ml — yes 0.330 1.650 1.00 3.5 − 10 ml  10 ml — yes 0.1100.550 0.30 1.1 − 10 ml  10 ml — no 30N-Rd2 1.000 5.000 32.50 —### + 2 ml10 ml — yes 0.330 1.650 22.00 3.7 + 2 ml 10 ml — no 0.110 0.550 9.003.1 + 2 ml 10 ml — no 30N-Rd3 0.330 1.650 34.00 13 + 2 ml 10 ml — no0.110 0.550 15.00 4.5 + 2 ml 10 ml — yes 0.033 0.165 6.20 3.5 + 2 ml 10ml — yes 30N-Rd4 0.330 1.650 15.25 25.0 + 2 ml 10 ml — — 0.110 0.5507.00 11.0 + 2 ml 10 ml — — 0.033 0.165 2.00 3.0 + 2 ml 10 ml — —¹Protein concentration in nanomolar²RNA concentration in nanomolar³Bound RNA expressed as % of max⁴Signal to noise⁵Use of nitrocellulose prefiltered RNA⁶Use of preblocked nitrocellulose with BSA⁷Volume in ml of buffer wash⁸Volume in ml of 0.5 M urea wash⁹Pool carried forward to the next round (comb. means that the pools werecombined before taken to the next round)¹⁰ Not determined

TABLE 7 SEQ ID NO: 5H7: 5′BBBccgcAUCGUCCUCCC 3′ 42 3H7:5′BBBUCGGGCGAGUCGtctg 3′ 43

B is biotin. A, G, C, and U are 2′-O-methyl-2′deoxy-ribo-adenosine,-guanosine, -cytidine, or -uridine, respectively, while a, g, c, and tare 2-deoxy-ribo-adenosine, -guanosine, -cytidine, or -thymidine,respectively. TABLE 8 Short PCR Templates and Primers SEQ ID NO:Starting DNA templates: 30NTR: 5′ GGGAG[-30N-] CGGGCGGG 3′ 44 30NTR: 5′GGGAG[-27N-] CGGGCGGG 3′ 45 SELEX PCR Primers: 5GTR: 5′TAATACGACTCACTATAGGGAG 3′ 46 3GTR: 5′ CCCGCCCG 3′ 47

TABLE 9 Sequence of isolated ligands from VEGF Rd1 Truncation SELEX byhybridization SEQ Ligand Sequence Phenotype ID NO: Family 1 13aUUGAAGAAUUGGGCGCAUGUUCUCCGUCCU 1 48 18 AAACGGAAGUAUUGGAUACAUAAGCACCCCU49 9 CAGGAUUUUGGAAGAAUUGGAUAUUGGCCU 2 50 20CUUAACUUUUGGAAGAAUUGAAUACUOGGU 4 51 10,16bUGAAACGGAAGAAUUGGAAACAUUGCUCGU 4 52 14a GAAACGGAAGAAUUGGAUACUCGCUGUGGU53 4b AGACUUUGGAAGAAUUGAAUUUGUCCGUGU 2 54 15b,19ACAUGUAGGAAGAAUUGGAAGAUGCCGCGU 2 55 5a UAGGAAGUAUUGUAAGUGUGUUGUCCUCGU 156 2b ggAAGAAUUGAUACGAUCGUCCAUCUACUCCU 1 57 Family 3 2a,23 (VT30.3)AGAAUCAGUGAAUGCUUAUAAAUCUCGUGU 4 58 3a (VT30.1)AACUAGUGAAUGCUUAUACGACCGUGUUGU 2 59 5b AAUCAGUGAACGCUUAUAGCUCUGCAUGGU 160 7 AUCAGUGAAUGCUUACAAACCCUGUGUCCC 1 61 22CUUUUUCUGAAUCAGUGAAUGCUUAGUGCU 1 62 Orphans 1AGCUAGGUGAAUGCCGAUAUUCUCUUCCGU 4 63 21b UACUAGGUGAAUGCCGAUAAUCUUAUCCGU 364 11 AUGOAAGUAUUGAGCCGAUUGUCAUCUCCC 1 65 15aUCUUUGGGUUUUUGCCAACGGUUUUCGCC 66 14b UCGAUCGCUUAUUUUCUCGGUCAUCCUCCC 674a AAACGGAACUUCUUGGAUACAUCUGCUCGU 3 68 3b UUGAAUAUUUCUCGCUCGUOAUUCCCGCCU5 69 6 AUUUGGAUGCAUGUCAAGGCGUUUUGCCCU 4 70 13b,21aUGUUGAUCGAGAUUUAAUCUAUUUCCACGU 3 71 16a UGAUCGAUUUCCUGGUCUGUUCUCCCUCCU 573 12 AUCAGUAUUGGCUGCUUCUAUUCCUCUGGU 74 24AAGGCCACUUGUAUGUGAUUCAGUAUUGGU 2 75Sequence shown is only from the random region of the molecules.Identical sequences are indicate by additional designation numbers.Ligands that were also found in the first SELEX experiment (Ruckman etal., J. Biol. Chem. 273:20556-67, 1998) are shown by paraenthases withtheir designation from the first SELEX experiment. Phenotypes wereclassified according to five groups as follows: (1) Molecules that losesignificant affinity for VEGF upon removal of their fixed sequences; (2)Molecules# with affinities for VEGF somewhat affected (positively or negatively)upon removal of their fixed sequences; (3) Molecules that gainsignificant affinity for VEGF upon removal of their fixed sequences; (4)Molecues with affinites for VEGE not affected upon removal of theirfixed sequences; and (5) Non-binding ligands either with or withoutfixed sequences.

TABLE 10 Sequence of isolated ligands from VEGF Rd1-ct Truncation SELEXby hybridization Ligand Sequence Phenotype SEQ ID NO: Family 1 34, 36aUGAAACGGAAGAAUUGGAAACAUUGCUCGU 1 52 31a AUUUGGAAGAAUUGGAUUUAGCACGUCCCU75 36b GAACGGAAGAAUUGGAUACGCUAGCAUGGU 76 38 UAAACGGAAGAAUUGGAACAUUGCUCGU4 77 44 CUUAAGUUUUGGAAGAAUUGAAUACUGGGU 2 51 Family 3 26AAACCAGUGAAUGCUUAUCGGAUCCGUUGU 4 78 27, 33AAAUCAGUGAAUGCUUAUAGUUUCUCGCGU 2 79 32 AAUCAGUGAAUGCUUAGAAAUCCACACCGU 280 39 AUCAGUGAAUGCUUACAAACCGUGUGUCCU 1 81 41aAAUCAGUGAAUGCUUAGAAAUCCACACCGU 82 45 GGAAAUCAGUGAAUGCUUAUACCUUCGCCU 1 83Orphans 25 AUAACAGAAUUUUUGGAGAACAAGUGUCGU 1 84 35AAAUUGACUAGUUUCGGUCUUCUACCCCCU 1 85 28 UUGAAAUUUCUCGGUCUUUCUCUCCCUCCU 186 29 UGUAGAGGUUUUGACUUUUCCCUUUUCCGU 2 87 31b, 41b,UUGACACUUCUCGAUUGUUCUCCUCUCCU 88 42b 48 UUGAUCGGACGUUAGUCAUUUCCCGAUCGU89 42a UUGAUCGACUUUCCUGAUCUUCUCCUCCU 90 43GAUCACGAACAUUUUGACGAUUUUCCUCCC 1 91 46 ACACUGGUUCCGAAGUAUUGUCUUUGUCCU 192 47 GGCUUAUUGGGCGUCAACAUUCUUUUCACGUC 93Sequence shown is only from the random region of the molecules.Identical sequences are indicate by additional designation numbers.Phenotypes were classified according to five groups as follows: (1)Molecules that lose significant affinity for VEGF upon removal of theirfixed sequences; (2) Molecules with aflinities for VEGF somewhataffected (positively or negatively) upon removal of their fixedsequences; (3) Molecules that gain significant affinity for VEGF uponremoval of their fixed# sequences; (4) Molecules with affinities for VEGF not affected uponremoval of their fixed sequences; and (5) Non-binding ligands eitherwith or without fixed sequences.

TABLE 11 Sequence of isolated ligands from TGFe1 Rd2 Truncation SELEX byhybridization SEQ Kd ID Ligand Sequence (Nm) Plateau NO: Family 1 *2, 7,44 GGUGCCUGUAGUCUUUGCAUCUUAUAAAUGCAAUCUGCCC 4.0 10% 94 *18GGUGCCUUUAGUCUUUGCAUCUUAUAAAUGCAAUCUGCCC 0.2  5% 95 Family 2 62UAGUGAUGAAUUUUUGCUGGAUCUGGUUUUGAACCGUCCC NB 96 20UAGUGACGAAUUUUUGCUGCAUCUGGUUUUGAACCGUCCC NB 97 3UAGUGAUGAACUUUUGCUGGAUCUGGUUUUGAGCCGUCCC NB 98 16UAGUGAUGAACUUUUGCUGGAU UGGUUUUGAACCGUCCC NB 99 Family 3 51AUCUGAAUUUAUUCGUCUACAGUUAC GCUGGGCCUUCCG 0.3 10% 100 41AUCUGAAUUUAUUCGUCUACAGUUACGGCUGGGCCUUCCG 0.6 10% 101 *1, 12AUCUGAAUUUAUUCGUCUACAGUUACAGCUGGGCCUUCCG 1.0 10% 102 Family 4 14, 43a,71 AUGCCUUUUGCCUUCAGGGUGUGAUUCCUUGAUCUGUCCG 0.6  5% 103 *70GUGCCUUUUGCCUAGGUUGUGAUUUGUAACCUUCUGCC 0.2 25% 104 Family 5 33UUAGUUCGGGCUCAACACCGCUAAUAUUCUUCGUUCCCC NB 105 22,28UUAGUUCGGGCUCAACACCGCUAAGAUUCUUCGUUCCCC NB 106 49UUAGGUCGGGCUCAACACCGCUAAAAAAUUCUUCGUUCCCC NB 107 Family 6 *29UGCCUUUAGUCUGAAUCUUACCAUGAUUCUCUGCCG 0.8 10% 108 39UGCCUU AGCAUGAAUAUACUGAUGUAUAUUCUCUGCCC NB 109 60UGCCUUUAGCCUG AUAUGCGUUUCGUGUAUAUCUCUGCCG 110 Family 7 50GACGUAGCGGGAUGCUUUAACUUUGAUCGUCCAUCAUGUG NB 111 53GAUGUAGCGGGAUGCUUUAACUUUGAUCGUCCACCAUGUG NB 112 Family 8 66AGUUUCAGGAUAUGUUGUGUGGUCGUUCUUUUUCCUCCC NB 113 32AUCUGGGUGACCUCUGUGUACGUUUAUUUUUACCGACCC NB 114 *56AAGGCAAGAAGCUUUAUGUGUCGCGUAACACAACUGUCCG 4.0 30% 115 40AGUUUUGGGAUCGCCACAGAUCUUACUGUGAGCUACUGUG 3.0  5% 116 *59UCUUUCGAACUGGGAAUUUUUGGUGUAGCCGUAUGCC 0.5 40% 117 *31AAGACCGUUCCGAGUGGUACAAGUAAACCCCUGUGUUCCG 2.0 60% 118 Family 9(nitrocellulose binders) 46 GUUUCUCUUUCACAUUUUUUUUUUUUUUUUUCACUUCCC 11954 GAUAGGUUUUUUUUCUAGGUUUUUUUUUUCAGUGUCCC 120 65CGUUGUUUUUUCUUUAUUUUUUGUUCUUUUGGUUGGC 121 61UGACCACAUUUAUUUUUUCUUCUUACCUCCUUUGGUCCC 122 63UCUUCAUCUGUCUUUUUAUCUCUCUCUUCUCACGCUCCC 123 48CCUAAGCUUCCUUUUAUUUUUUUCUUCUUUAAUUUCCUGGGC 124 6CUCUUUUCUUUAUGUUUUUUUCUUUUUUUCUUGUCCCCC 125 13UCCCAUCAUCCAAGCGUGAUACUUUUUUUUUCCCCUCCC 126 69GACCUUUUUUUCUUGCUUUUCUUUUUGCCUUUCCGUCCC 127 19UUUCGUUUUCUUUAUCUUUUUUCUCGUUUUUUGCCCC 128 30UUAAUUUCAUAUUUUUUUUUUUCUUUUUCCCUAACGUGGC 129 36UCCCUAUCACAACUUUGUUUUCUUUUAUUUUUCUCUUCGC 130 38GNNCUGGGUUCNACUUUNCAUAUUUGUNUUUUUU 131 24GACGUUGUGUUUACUGAUUUCUUUUUCUUUUUUCCGCCUG 132 37CACUAGUCAUUUUCUAUCUUUCUUUUUCUCCCUUGUGCCC 133 25ACUGGGUUUAUUCUUCUUUUUUCUUGUUCCUACCACCCCC 134 45AUCCUCUUGUCAUAGAUCGUUUGUUUUGUUUUUGUACCG 135 10UCUUUUCUCUGUUUCCUUUUGUUUUUCCCUGUACUCCC 136 58UCCUUUGGUUUUAGUUGUUAUUGUUUUUCCUUUUGUGUCGC 137 57CGACCAUUUAUUUCUCUUAUCAUUCUUUUCUCCCUAUCGC 138 15UCGUCGGAUUCUCUAUGUUUUGUUUUCAUUUCUUCCCCC 139 73UCGAACUAUUACUCUUUUAUUAUUCCUUAAUUUUUGCCGC 140 4AUUGAGGGUUUCUUUUUCGUCUUUUUCCUUUCCCUCUCCC 141 47UUCCGGUCUUUUCUUGUGUUUAUGUUUCUUUCUGUUGCC 142 5GGACAUAUUUUCUUCUUCUUCCUCUGCUUUUUGUUGUCCC 143 55GUACUUGCUUCUCUACUAUUUUCUCCUCAUUCCCCUGUG 144 72UUCUUCGUUUCUUCUCUCUCUUCUAGCCGUCCUUCGCCCC 145 64CUCAGUUUAUAUGACACUUCACUUUCUUUUCGUUUUACCG 146 42UGCGACAUUAUUUAAUUUUCUCCUUCCUUUCAUCGUGCC 147 Orphans 34CAGCUCACUUAUAUUUCCGUCCAAUUCCUUCUUUACUGCC NB 148 *52UGUCUUUAGCCUACAGUUGACUGUUCAAUUGUUCUGCCG 1.0 30% 149 23UGUUUGUGCUACGACCUACAUUCGUUGGAAUGUUCUGCCG 0.4 10% 150 *67AUCACUAGGCUCAUUUGUGAGCCGUUAUUCCUUGACUC 0.1  8% 151 11AGUGAAUUGCAUCCUUCGAUUACCUACUCUUUUGUGCCC NB 152 43bGGAGGGAAAUGAAAUGACAAGAACGAGACUAAGAUGGGA 153 27 UUGUUCCG 154 35NUCUUNUUCCCUCNANUGUCCC 155 26 CNUAA 156 68 GGUGUNUUU 157Sequence shown is only from the random region of the molecules.Identical sequences are indicate by additional designation numbers.Ligand affinities were determined by nitrocellulose filter bindingfollowing removal of fixed sequences and are indicated by Kd andplateaue values obtained. NB indincate ligands unable to bind the targetunder the experimental conditions used. Stars (*) indicates repeats ofbinding experiments where we are showing the best determined values forKds and plateaues.

TABLE 12 Sequence of Rd12 VT30 ligands tested for binding without fixedsequences SEQ Sequence of Random Region ID NO: Ligand 5′gggaggacgaugcgg[Random Region] cagacgacucgcccga Phenotype 158 Family 1 VT30.20AAACGGAAGAAUUGGAUACCGCUACGUGUU 1 159 VT30.7UAACCAGUGGAAGAAUUGGCUGCUAUCCU 2 160 VT30.4CUUAAGUUUUGGAAGAAUUGAAUACUGCGU 1 161 VT30.53AGCUAACGGAAGAAUUGGAAACAACCGCGU 2 162 Family 2 VT30. 9, 14, 34,UCAACCGGUUGAAUAUUUGGUCGCUGACCU 4 163 37, 48, 50 Family 3 VT30.1, 25AACUAGUGAAUGCUUAUACGACCGUGUUGU 1 164 VT30.21AUCAGUGAAUGCUUAUAGACCGUAUUGCGU 1 165 VT30. 3, 5, 16,AGAAUCAGUGAAUGCUUAUAAAUCUCGUGU 2 166 31, 36, 43 VT30.44cggAAUCAGUGAAUGCUUAUACAUCCGCUCGGU 1 167 VT30.15AACCAGUGAAUGCUUAUAAGACUGCUCGU 1 168 VT30.29AAUCAGUGAAUGCUUAUAGCUCCGCGUGGU 1 169 VT30.35ACCAGUGAAUGCUUAUAAGCCCAUCGACCU 1 170 VT30.45AAUCAGUGAAUGCUUAUAGCUCCGNGUCCU 1 171 Orphans VT30.12,UCUUUGGGUUUUUGCCAACGGUUUUCGCU 2 172 30 ,39, 46 VT30.40AUUUGGAUGCAUGUCAAGGCGUUUUGCCCU 2 173Sequence information shown is from published work (Ruckman et al., J.Biol. Chem. 273:20556-67, 1998). Sequence shown is only from the randomregion of the molecules except for VT30.44 where the required forbinding portion of the 5′ fixed sequence is also shown in lower case.Identical sequences are indicated by additional designation numbers.Phenotypes were determined by filter binding and were classifiedaccording to five groups as follows: (1) molecules that lose significant# affinity for VEGF upon removal of their fixed sequences; (2) moleculeswith affinities for VEGF somewhat affected (positively or negatively)upon removal of their fixed sequences; (3) molecules that gainsignificant affinity for VEGF upon removal of their fixed sequences; (4)molecules with affinites for VEGF not affected upon removal of theirfixed sequences; and (5) Non-binding ligands either with or withoutfixed sequences.

TABLE 13 SEQ ID NO: 3G7RC 5′ pCAGACGACTCGCCCGABBB 174 3G7RCD 5′pGACGACTCGCCCGABBB 175 3G7R6 5′ TCGGGCGACTCGTCTCNNNNNN 176 3LBsmC 5′pCGCATTCTCCCTTTABBB 177 3LBsm 5′ GGAGAATGCGNNNNNN 178

TABLE 14 Sequence of isolated ligands from VEGF 5′ Truncated StartingPool SEQ Sequence of Random Region ID NO: Ligand 5′gggag [Random Region]cagacgacucgcccga Phenotype 179 Family 1 T1-26UGUGAAACGGAAGAAUUGGAAACAUUGCUCCUCA 180 T1-19CUGCCUUAAGUUUUGGAAGAAUUGAAUACUGGGUCA 4 181 T1-47UGGAAGAAUUGGAUAUAUCGUUCGUUUUCCGGUCA 182 T1-21UUGGAAGAAUUGAUACGAUCCUCCAUCUACUCUUCAG 1 183 T1-11GAAGAAUUGGAAACAUUGCUCGU 5 184 Family 2 T1-22UGGUCAACCGGUUGAAUAUUUCGUCGCUGACCUA 1 185 Family 3 T1-49CGGAAAUUAGUGAAUGCUUAUAACUUCCACGGUCA 186 T1-7GGACUAGGUGAAUGCCGUUAUUCUUCCUGUCA 3 187 T1-30GCGGACUAGGUGAAUGCCAAUAUUCUUCUCCGUC 188 T1-39-1GGGGACUAGGUGAAUGCCAAUAUUCUUCUCCGUCA 189 T1-50CUAGGGACUAGGUGAAUGCCAAUAUUCUUCUCCGUCA* 190 T1-1CGGACGACCUGGUGAAUGCCAAUAUACUUUUCGCGUCA 5 191 T1-38GCCCUCAGCUACUCGGUGAAUGCCAUUAUGCUUGCCCU 192 T1-15GGGGCGGGAGUGAAUGCUUAUUAGAUCUGCCGUCA 5 193 T1-5GGUGAAUGCCAACCAUUUUAUCGCCUAUCGUCA 5 194 T1-42-1CUGGUGAAUGCCAACGAUUUUAUCGCCUAUC 195 Family 4 T1-13CAGACGACUCGCCCGACAGACCACUCGCCCGA* 5 196 T1-3CAGACGACUCGCCCGAGGAGGAGGGGGC* 5 197 T1-2ACCACCAGACGACUCGCCCGAACGCUUAUCCUCUGGU* 5 198 T1-4GCAGACGACUCGCCCGAGGAUACACAUCGUGUGGU* 5 199 T1-29AUAGGCAGACGACUCGCCCGAGGAAACAUUGCUCCU* 200 T1-41CAGACGACUCGCCCGACAGACAACUCCCCC* 201 T1-53GCAGACGACUCGCCCGAUUGAAUUUGUCCGUGU* 202 T1-54 CAGACGACUCGCCCGACAGACGACUC*203 T1-44 UGCAUCAGACGACUCGCCCGACAACACUCGCCCGA* 204 T1-55GGGGCCAGACGACUCGCCCGACAGACGACUC* 205 T1-42-2NGCAGCAGACGACUCGCCCGACAGACGAUCGCCCGA 206 Orphans T1-9GUCUUCGAAUCAGUAAAUCCUUAGCGCUCGU* 1 207 T1-17AGAGGUUUCAGUAUUGGCAUCGCGUUUGUCCUCA* 1 208 T1-18CUAGUUGAUCGAUUUCCUGAUGUCCUUUCCUCCUCA* 1 209 T1-23GAAUUGGAUACUCGCUGUGGUUCUUCCCCCU* 210 T1-37GCAUUGACUAGGCUAGGCUUCUCUUUCCCCA* 211 T1-25UGAUCAAAAGUGGAUUCUUCGUUUUUCCCCCCCA* 212 T1-28, 31UAGUUGACUUUUCCCGAUUAUCCUCUCGUGCCUCA* 213 T1-36GUGGACACUGGUUCCGAAGUAUUGUCUUUGUCCU* 214 T1-39-2CGUGACAUUUCUCGAUCGUAAUACCUCCCCCU 215 T1-43 UUCCGCUUUGGCAUUCUUCGUCUCCUCA*216 T1-48 UGGUUGGCACUUCUCGAUUGUUCUCCUGUCCUCA* 217 T1-35CAGGUGUNGCACAUGGUGCUG* 218 T1-40 GGGGAGGACGAUGCG 219 T1-16 GUCUC* 5 220T1-34 CGCUG* 221Sequence shown is only from the random region of the molecules. Allligands start with 5′GGGAG except T1-37 and T1-35 which start with5′GGGAT and 5′GGGA, respectively. Identical sequences are indicate byadditional designation numbers. Phenotypes were classified according tofive groups as follows: (1) Molecules that lose significant affinity forVEGF upon removal of their fixed sequences; (2) Molecules withaffinities for VEGF somewhat affected (positively or negatively) uponremoval of# their fixed sequences; (3) Molecules that gain significant affinityfor VEGF upon removal of their fixed sequences; (4) Molecules withaffinities for VEGF not affected upon removal of their fixed sequences;and (5) Non-binding ligands either with or without fixed sequences.Stars (*) indicate repeats of binding experiments.

TABLE 15 Sequence of isolated ligands from VEGF Rd1 Truncation SELEX byLigation SEQ Sequence of Random Region ID NO: Ligand 5′gggag [RandomRegion] cagacgacucgcccga Phenotype 179 Family 1 T2-2CUGGAAACGGAAGUAUUGGAUACAUAAGCAUCCCCACA 2 222 T2-6GGAGACUUUGGAAGAAUUGAAUUUGUCCGUGUCACA 3 223 T2-7GAAACGGAAGAAUUGGAAAACACCCGUC 2 224 T2-14GGAGACUUUGGAAGAAUUGAAUUUGUCCGCGUCA 225 T2-20GGGAGGAAACGGAAGAAUUGGAAAACACCCGUCAGCA 2 226 T2-21GGUGAUGCAGUGGAAGAAUUGGUUGCAGCCGUCACA 3 227 T2-24-1UCGUGAAACGGAAGAAUUGGAAACAUUGCUCGUCCA 228 T2-24-3GGCUAGUAGGAAGAAUUGUAAGCUGCCUCGUGCA 229 Family 2 T2-9CGGGGAUAACAGAAUUCUUGCUGAACAACCGGUCACA 3 230 T2-5UGGUCAACCGGUUGAAUAUUUGGUCGCUGACCUCA 3 231 T2-24-2UGGUCAACCGGUUGAAUAUUUGGUCGCAGAC 232 T2-16GGGUCAACCGGUUGAAUAUUUGGUCGCUGACCUCACA 233 Family 3 T2-1GGCGAAUCAGUGAAUGCUUAAUGCUCCUCGGUCACA 3 234 T2-4UCUCGAGAAUCAGUGAAUGCUUAUAAAUCUGUGUCCA 3 235 (VT30.3) T2-10GGGACCGGGUGAAUGCCAAUGUACUUUUCGCGUCCA 3 236 T2-15GGUACCUAGGUGAAUGCCGUUAUUCUGUUGCCCACA 3 237 T2-17AGGAGAAUCAGUGAAUGCUUAUAAAUCUCGUGUCACA 2 238 (VT30.3) T2-18UGGAAAUCAGUGAAUGCUUAUAGUUUCUCGCGUCACA 4 239 T2-23GGACUGAAUGAAUGUUGACGGUUACGCUUUCCCCA 4 240 Orphans T2-11GGACACUGGUUCCGAAGUAUUGUCUUUGUCCUCACAG 2 241 T2-12GGGACACUGGUUCCCAAGUAUUGUCUUUGUCCUCACA 3 242 T2-22GGACACUGGUUCCGAAGUAUUGUCUUUGUCCUCACA 3 243Sequence shown is only from the random region of the molecules.Identical sequences are indicate by additional designation numbers.Ligands that were also found in the first SELEX experiment (Ruckman etal., .1. Biol. Chem. 273:20556-67, 1998) are shown by parentheses withtheir designation from the first SELEX experiment. Phenotypes weredetermined by filter binding and were classified according to fivegroups as follows: (1) Molecules that lose significant affinity for VEGFupon removal of theirfixed sequences; (2) Molecules with affinities for VEGF somewhataffected (positively or negatively) upon removal of their fixedsequences; (3) Molecules that gain significant affinity for VEGF uponremoval of their fixed sequences; (4) Molecules with affinities for VEGFnot affected upon removal of their fixed sequences; and (5) Non-bindingligands either with or without fixed sequences. Stars (*) indicaterepeats of binding experiments.

TABLE 16 Sequence of isolated ligands from VEGF Rd2 Truncation SELEX byLigation SEQ Sequence of Random Region ID NO: Ligand 5′gggag [RandomRegion] cagacgacucgcccga Phenotype 179 Family 1 T3-5GGUUUGGAACGGAAGAAUUGGAUACGCACCUCACACA 3 244 T3-13AGGAUGUAGGAAGAAUUGGAAGAUCCGUCUGCGUA 3 245 T3-15-2UGGCAGGAUUUUGGAAGAAUUGGAUAUUGGCCUCA 246 T3-17-1GGACCUUUUGGAAGUUAUUGGAUAGGCCGUCUCACGCA 247 T3-18GGACAUGUAGGAAGAAUUGGAAGAUGCGCCACAG 2 248 Family 2 T3-2GGUAACCGGUUGAAGUUAUUGGUCGCUAUGCU 3 249 T3-6GGAGGUCAACCGGUUGAAUAUUUGGUCGCUGACCUCACACA 250 T3-8GGUCAACCGGUUGAAUAUUUGGUCGCUGAUCUCGCCACA 3 251 T3-9UGGUCAACCGGUUGAAUAUUUGGUCGCUGACCUCACA 3 252 T3-11AGCGUAACCGUCAACAUUCAUUCAGUCCCCUCCC 5 253 FAMILY 3 T3-7GGAGAAAUCAGUGAAUGCUUAUUGCUUCUCGUCACACA 3 254 T3-10GGCACUAGGUGAAUGCCGUUAUUCUUGCUGCUCUCUCU 2 255 T3-12-1GGGAGAAAUCAGUGAAUGCUUAUCGUUUCUCGUCACACA 256 T3-12-2GGGGACUAGGUGAAUGCCAAUAUUCUUCUCCGUCACCACA 257 T3-14GGCGGAUCAGUGAAUGCUUACAAACCGUCUGUCCC 2 258 T3-15-1GCAAAAUCAGUGAAUGCUUAUUGCUUUGGCUCACCA 259 T3-16GGCGGAACUAGUGAAUGCUUAUACGACCGUCUUGUCACA 4 260 T3-19UUAGGAAUCAGUGAAUGCUUAUACAUCCGCUCGGUC 3 261 Orphans T3-1UGGACACUGGUUCCGAAGUAACGUUGAAGUAAAAUUCGUUCUCUCGGCGUUUGGC 2 262 T3-3GGACACUAGGUGCAUGCCAAAAUUCUUGUCCUCAGCA 2 263 T3-17-2GGGACAUUGGUUCCCkAGUAUUGACUUUGUCCUCACACA 264Sequence shown is only from the random region of the molecules. Allligands start with 5′GGGAG except T3-11 which starts with 5′GGGAA.Identical sequences are indicate by additional designation numbers.Ligands that were also found in the first SELEX experiment (Ruckman etal., J. Biol. Chem. 273:20556-67, 1998) are shown by parentheses withtheir designation from the first SELEX experiment. Phenotypes weredetermined by filter binding and were classified according to fivegroups# as follows: (1) Molecules that lose significant affinity for VEGF uponremoval of their fixed sequences; (2) Molecules with affinities for VEGFsomewhat affected (positively or negatively) upon removal of their fixedsequences; (3) Molecules that gain significant affinity for VEGF uponremoval of their fixed sequences; (4) Molecules with affinities for VEGFnot affected upon removal of their fixed sequences; and (5) Non-bindingligands either with or without fixed sequences. Stars (*) indicaterepeats of # binding experiments.

TABLE 17 Sequence of isolated ligands from 30N TGF

1 Rd2 Truncation SELEX by Ligation SEQ Sequence of Random Region ID NO:Ligand 5′gggag [Random Region] cagacgacucgcccga Kd (Nm) Plateau 179Class 1 30NtruNc 1, 11, GGUUAACCGUUAAGACGGCGUCAUUUUGUCCC 0.081 30% 26522, 26, 29, 30, 31, 33 30NtruNc 8, 40 GGUUAACCGUUAAGACGGCUUCAUUUUGUCCCNB 266 30NtruNc 24, 25 GGUUAACCGUUAAGACGGCGUUAUUUUGUCCC 2.5 30% 26730NtruNc 10 GGUUAACCGUUAAGACGGCNUCAUUUUGUCCC NB 268 30NtruNc 13GGUUAACCGUUAAGACGGCUUUAUUUUGUCCC 1.4 20% 269 30NtruNc 7GGUUAACCGUUAAGACGGCGUNAUUUUGUCCC 2.0 25% 270 30NtruNc 36GGUUAACCGUAAAGACGGCAUUAUGUAGUCCC 31.0 40% 271 Class 2 30NtruNc 20, 14GGGAAUUUUUGGUAAAGCCGUAUGCCUCGC 0.34 25% 272 30NtruNc 27GGGAAUUUUUGGUAAAGCCAUAUGCCUCGCCA >30.0 273 30NtruNc 19CGGGAAUUUUUGGUAAAGCCGUAUGCCUCGCCA 0.56 35% 274 30NtruNc 12UGGGAAUUUUUGGUAAAGCCGUAUGCCUCGC 0.88 35% 275 30NtruNc 34CGGGGAAUUUUUGGUAAAGCCGUAUGCCUCGC 276 30NtruNc 3GGUUUCAUGGAAUUUUUGGUAAAGCCGUAUGCCUCGCCA 30.0 70% 277 30NtruNc 18AUUUUUGGUAAAGC NB 278 30NtruNc 38 GGAAUUUUUGAUUUAGUCGUACGCCGCAUCCC 0.217% 279 30NtruNc 6 GGUUUCAUGGAAUUUUUGGUUUAGCCGUAUGC 2.0 35% 280 30NtruNc2 GGUUCUGGAAUUUUUGGUUUAGCCGUACGC 1.2 37% 281 30NtruNc 4AGGGAUCUGGAAUUUUUGGUUUAGCCGUACGC 0.088 20% 282 30NtruNc 32ANCUGGUAAUUUUGGUUUANCCGUAUNCC 22.0 15% 283 30NtruNc 9AGGGGUCUGGAAUUUUUGGNUUACCCCUACGC 0.1 20% 284 30NtruNc 23GGAAUUUUUGUGUAGACGUAUGCCCUUUGCC 0.93 35% 285 30NtruNc 37CGGAAUUUUUGUGUAGACGUAUGCCGCUUUGNC 0.083 15% 286 Class 3 (nitrocellulosebinders) 30NtruNc 15 GGGCUCAACUUUUCUCUUCUUCUUUUCCGCCC 287 30NtruNc 5GGCCCCAUUCUUUUUUAUUUCUUUUUUGCCCCA 288 30NtruNc 2GGCCCCGGUUUUUCUUUUUCUUUUCUUUUUCCC 289 30NtruNc 39GGCCUUCUUUCUUUCUUUUCUUUUUUCCGUCCC 290Sequence shown is only from the random region of the molecules. Allligands start with 5′GGGAG except 30NtruncNc-9 and 30NtruncNc-18 whichstail with 5′GGGTC and GGGA, respectively. Identical sequences areindicate by additional designation numbers. Ligand affinities weredetermined by nitrocellulose filter binding following removal of fixedsequences and are indicated by Kd and plateau values obtained. NBindicate ligands unable to bind the target under the experimentalconditions used.

TABLE 18 Sequence of isolated ligands from 40N TGFβ1 Rd2 TruncationSELEX by Ligation SEQ Sequence of 5′Fixed-Random Region ID NO: Ligand5′gggag [Random Region] cagacgacucgcccga Kd (nM) Plateau 291 Family 1AG34 GGGA           CAGACGACUCGCCCGA    UAAUACGACUCACUAUAGGGAGGN 292AG30 GGGAG        GGCAGACGACUCGCCCGA    UAAUACGACUCACUAUA 293 AG22GGGA          CACGACGACUCGCCCGA    UAAUACGACUCACUAUAGGNAGUUG 294 AG8GGGA  CUCACUAUACAGACGACUCGCCCGA    CCGCUAUUACAAUCUUCGCCUCCC 295 AG15GGGA          CCAGACGACUCGCCCGA    GGGAACGUUCUCCCACCUUCCUGCC 296 AG7GGGA          CAGACGACUCGCCCGA    GGGAUUUUACGUUCGUCUCGCGUCUCCC NB 297Family 2 AG6GGGAG          CAGACGACUCGCCCGA  ACUGGGAAUUUUUGGUUGAGCCGUAUGCC 0.004 12%298 (0.2) (25%) AG24GGGAG          CAGACGACUCGCCCGA  GCUGGGAAUUUUUGGCUGAGCCGUAUGCC 299 AG48GGGAG      GGUCCAGACGACUCGCCCGA   GGGGGAAUUUUUGGUUGAGCCGUAUGCC 300 AG4GGGAG          CAGACGACUCGCCCGA    GGGGAAUUUUUGGUUGAGCCGUAUGCC 301 AG31GGGA           CAGACGACUCGCCCGA GACUGGGAAUUUUUGGUUGAGCCGUAUGCC 302 AG40GGGA           CAGACGAACUCGCC CGAACUGGGAAUUUU GGUUGAGCNGUAUGCC 303 AG16GGGAG                   GGUGUUUCGAACUGGGAAUUUUUGGUUUAGCCGUAUGCC 0.02 23%304 AG13,GGGAG                   GGUGUUUCGAACUGGGAAUUUUUGGUUGAGCCGUAUGCC 305 27,33 AG35 GGGAG                    GGUGUUUCGAACUGGGAAUUUUUGGUUGAGCCGUAUCCC306 Family 3 AG11 GGGAG GGAUUCUGCCGAGUUAAUUUCGGUGUCUGUAGCUUAUCCC 1.0 22%307 AG2 GGGAG GGAUUCUGCCGAGUUAACGUCGGUGUCUGUAGCUUAUCCC 1.0 25% 308Family 4 AG32 GGGAG GACGAUGCGGGGGUUAUUGGGNGUCAACAUCCCCGAUUCUUUUCACGUC309 AG36 GGGAG GACGAUGCGGGGGUUAUUGGGCGUCAACAUCCCCGAUUCUUUUCACGUC 310 AG9GGGAG GACGAUGCGGGGGUUAUUGGGCGUCAACAUCCCCGAUUCUUUUCACCUC 311 Family 5AG21 GGGAG GACGAUGCGGAGCGGAUUAAUUAGUCUGACUUCUUGUCCC 312 AG25GGGAG GACGAUGCGGUAAAGUAGCAUUAUCCUCUAACAUCCUGCC 313 Family 6 AG18GGGAG GGGUGCCUUUAGCUUGGUCUGUUUAGUACAUUCCUCUGCCC 1.0 22% 314 AG1GGGAG GGGUGCCUGUAGUCUUUGCAUCUUAUAAAUGCAAUCUGCCC 0.1 14% 315 (0.5) (25%)Family 7 (Nitrocellulose binders) AG5 GGGAGG 316 AG29GGGAG GGUGUUACGAGCGUCGGACCCUGUUUCCAACAUCCUCCC 317 AG14GGGA  CUUCCUCCGGCUAUCAUUUUCUUCUCUUUCUCUCUCCC 318 AG12GGGAG GGAGUGGUCGACCAUGAUUCUUUUUAUUUCUCCUUCCUCCC 319 AG19, 20GGGAG GGACCUUUCUACUUCAUCAUUUUUCUUCACUCUCUCCGUCCC 320 AG37GGUAG GGAGGUUUCUGACUCUNAGCUUUCCUUCCUCCUGCCUCCC 321 AG38GGGA  CAGACGACUNGCCCGACGUAUGCC 322Sequence shown is from the initiator sequence (usually 5′GGGAG) andrandom region of the molecules. Identical sequences are indicate byadditional designation numbers. Ligand affinities were determined bynitrocellulose filter binding following removal of fixed sequences andare indicated by Kd and plateau values obtained. For biphasic binding,low affinity Kd and plateau values are in parentheses. NB indicateligands unable to bind the target under the experimental conditionsused.

1-10. (canceled)
 11. A method for identifying nucleic acid ligands of atarget compound, said method comprising: a) providing an candidatemixture of nucleic acids of differing sequences comprising randomizedsequences, b) contacting said candidate mixture with a target underconditions favorable for binding between the target and members of thecandidate mixture; c) partitioning those nucleic acids with higheraffinity for the target from those nucleic acids with lesser affinity tothe target to generate an enriched candidate mixture; d) ligatingnucleic acids in said enriched candidate mixture to a 3′ primer bindingsite using RNA ligase to generate a ligation product; e) reversetranscribing the ligation product using the 3′ primer to generate cDNA;f) ligating the cDNA to a DNA oligonucleotide encoding the T7 promoterto generate a new dsDNA candidate mixture; g) transcribing the newcandidate mixture to generate a new RNA candidate mixture; h) removingthe 3′ primer binding site to yield a ligand-enriched mixture of nucleicacids, whereby nucleic acid ligands of the target compound may beidentified.
 12. The method of claim 11 further comprising the step: i)repeating steps b) through h) using the ligand enriched mixture of eachsuccessive repeat as many times as required to yield a desired level ofligand enrichment, whereby nucleic acid ligands of the target compoundmay be identified.
 13. The method of claim 11 wherein said candidatemixture is prepared from a template comprising a region of conservedsequence and a region of randomized sequence.
 14. The method of claim13, wherein said conserved sequence is a restriction site, and whereinsaid preparation comprises digesting the candidate mixture with arestriction enzyme and transcribing the digested candidate mixture. 15.The method of claim 11 wherein said candidate mixture is prepared byproviding a candidate mixture of nucleic acids of differing sequencescomprising 3′-region of conserved sequence, 5′-region of conservedsequence, and a region of randomized sequence, and removing the3′-region of conserved sequence and the 5′-region of conserved sequenceby enzymatic digestion.
 16. The method of claim 15, comprising: a)providing an RNA candidate mixture comprising a 3′-region of conservedsequence, a 5′-region of conserved sequence and a region of randomizedsequence; b) generating a cDNA candidate mixture from the RNA candidatemixture; c) transcribing the cDNA candidate mixture to generate an RNAtranscript; d) removing the 5′ region of conserved sequence with RNaseHto generate digested RNA; e) reverse transcribing the digested RNA togenerate cDNA; f) optionally ligating a 3′-primer binding site to thecDNA; g) ligating the generated cDNA to a DNA oligonucleotide encodingthe T7 promoter-initiator to generate a ligation product; h) amplifyingthe ligation product to generate a new dsDNA candidate mixture lackingat least part of the 5′-region of conserved sequence; i) transcribingthe dsDNA to generate a 5′-truncated transcript; and j) digesting the3′end of the 5′-truncated transcript to generate a truncated RNAcandidate mixture.
 17. The method of claim 16, wherein the 3′-region ofconserved sequence comprises a primer binding site.
 18. The method ofclaim 17, wherein step d) further comprises removing the 3′ region ofconserved sequence with RNaseH.